Interaction of Myocilin with γ-Synuclein Affects Its Secretion and Aggregation

Cellular and Molecular Neurobiology, Vol. 25, No. 6, September 2005 ( C©2005)DOI: 10.1007/s10571-005-8471-4

Interaction of Myocilin with γ-Synuclein Affects ItsSecretion and Aggregation

Irina Surgucheva,1,4 Bum-Chan Park,2 Beatrice Y. J. T. Yue,2

Stanislav Tomarev,3 and Andrei Surguchov1,4,5

Received February 14, 2005; accepted April 12, 2005

SUMMARY

Mutations in the gene encoding human myocilin are associated with some cases ofjuvenile and early-onset glaucoma. Glaucomatous mutations prevent myocilin from beingsecreted. The analysis of the defects associated with mutations point to the existence of fac-tor(s) in addition to mutations that might be implicated in the development of glaucoma.In the present paper, we found that interaction of myocilin with one of the members ofthe synuclein family alters its properties, including its ability to be secreted. Results of im-munoprecipitation show that myocilin is a γ-synuclein-interacting protein. Further analysisdemonstrated that both myocilin and γ-synuclein are expressed in human TM cells, immor-talized rat ganglion (RGC-5) cells, and HT22 hippocampal neurons. According to Westernblotting, in addition to monomeric form with molecular weight 17 kDa γ-synuclein is presentas higher molecular weight forms (∼35 and 68 KDa), presumably dimer and tetramer.Myocilin and γ-synuclein have partially overlapping perinuclear localization. Dexametha-sone upregulates myocilin expression in RGC-5 cells and HT22 hippocampal neurons. Wefound alterations of myocilin properties as a result of its interaction with γ-synuclein. In cul-tured cells, γ-synuclein upregulates myocilin expression, inhibits its secretion and preventsthe formation of high molecular weight forms of myocilin. Although both α-synuclein andγ-synuclein are expressed in HTM cells, only γ-synuclein interacts with myocilin and altersits properties.

We conclude that myocilin and γ-synuclein interact and as a result, myocilin’s prop-erties are changed. Since myocilin and γ-synuclein have partially overlapping intracellular

1 Retinal Disease Research Laboratory, Veterans Administration Medical Center, 4801 Linwood Blvd,Kansas City, MO 66148, USA.

2 Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA.3 Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, MD, USA.4 Department of Neurology, Kansas University Medical Center, Kansas City, KS, USA.5 To whom correspondence should be addressed at Retinal Disease Research Laboratory, Veter-

ans Administration Medical Center, 4801 Linwood Blvd, Kansas City, MO 66148, USA; e-mail:[email protected].

Abbreviations used: AA, amino acids; CNS, central nervous system; co-IP, co-immunoprecipitation;DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HTM, human trabecular mesh-work; ICC, immunocytochemistry; IOP, intraocular pressure; IP, immunoprecipitation; ONH, opticnerve head; PA, paraformaldehyde; PBS, phosphate buffered saline; PCR, polymerase chain reaction;PD, Parkinson’s disease; POAG, primary open-angle glaucoma; RGC, retinal ganglion cells; RT, roomtemperature; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

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1010 Surgucheva, Park, Yue, Tomarev, and Surguchov

localization in cell types that are implicated in glaucoma development, their interactionmay play an important role in glaucoma.

KEY WORDS: myocilin; synuclein; glaucoma; ganglion cells.

INTRODUCTION

The glaucoma is a heterogeneous group of optic neuropathies characterized by pro-gressive loss of retinal ganglion cells (RGC), excavation of the optic nerve head(ONH), and visual field changes. Glaucoma is the second leading cause of irre-versible blindness worldwide affecting more than 66 million people (Quigley, 1996).There is evidence supporting the existence of a genetic component in several formsof glaucoma, including primary open-angle glaucoma (POAG) (for reviews see:Friedman and Walter, 1999; Johnson et al., 1996; Tamm, 2002; WuDunn, 2002).

Myocilin was implicated as a candidate gene in the cause of glaucoma. A numberof mutations were found to associate with autosomal dominant juvenile open-angleglaucoma and adult onset POAG (Adam et al., 1997; Stone et al., 1997; Alward et al.,1998; Michels-Rautenstrauss et al., 1998; Allingham et al., 1999; Fingert et al., 1999).Myocilin is a 504 amino acid protein with a predicted molecular weight of 55–57 kDa.Myocilin is homologous to several proteins including olfactomedin (amino acids 324–502) and leucine zipper motif within the N-terminal α-helix domain (Tamm, 2002).N-terminal domain of myocilin (amino acids 72–179) also contains a myosin-likeregion. More than 50 different mutations have been described in the myocilin gene(Fingert et al., 1999; Jacobson et al., 2001; Alward et al., 2002; Michels-Rautenstrausset al., 2002; Pang et al., 2002; Gobeil et al., 2004; Mackey et al., 1999). The detailedanalysis of the defects associated with mutations points to the existence of factor(s) inaddition to mutations that may be implicated in the development of glaucoma. Theidentification and characterization of this factor(s), including proteins interactingwith myocilin could lead to a breakthrough in elucidation of its role in the glaucomapathology (Wentz-Hunter et al., 2002).

Myocilin is expressed in different parts of the eye as well as in non-ocular cellsand tissues (Swiderski et al., 1999; Kulkarni et al., 2000; Noda et al., 2000; O’Brienet al., 2000; Tamm, 2002; Torrado et al., 2002; Jurynec et al., 2003; Ohlmann et al., 2003;Wentz-Hunter et al., 2003, 2004). It is related to a group of olfactomedin-containingproteins—secreted glycoproteins with conserved C-terminal motifs. Olfactomedinwas originally identified as the major component of the mucus layer that sur-rounds the chemosensory dendrites of olfactory neurons. Homologues were sub-sequently found also in other tissues, including the brain and in species ranging fromCaenorhabditis elegans to Homo sapiens. The expression of several olfactomedin-related proteins in mucus-lined tissues suggests that they play an important role inregulating physical properties of the extracellular environment (Kulkarni et al., 2000).

Depending on the experimental conditions of Western blot analyses, humanmyocilin can be detected as a major band at around 55–57 kDa, or this band may beresolved in a doublet (Nguyen et al., 1998; Caballero et al., 2000; Clark et al., 2001).Myocilin from mouse and some other mammalian species is 14 amino acids shorterat the N-terminus (Shepard et al., 2003). Recombinant myocilin, as well as secretedmyocilin in the aqueous humor and human trabecular meshwork (HTM) cell culture

Myocilin-γSynuclein Interaction 1011

supernatant, can form high molecular weight aggregates with molecular size of 120–200 kDa and higher (Nguyen et al., 1998; Caballero et al., 2000; Jacobson et al.,2001; Fautsch et al., 2004). Myocilin is localized in various organelles and accordingto several publications may be associated with the centrosome in some cell types(Noda et al., 2000; O’Brien et al., 2000; Ueda et al., 2000). Despite extensive efforts,the function of myocilin and its role in glaucoma remain elusive. It is believed thatmisfolding, abnormal aggregation, and defects in secretion play an important role inthe involvement of myocilin in glaucoma (Caballero et al., 2000; Jacobson et al., 2001;Fautsch et al., 2004; Liu and Vollrath, 2004). Recent evidence has shown that myocilinmutations contribute to glaucomatous alterations by acting intracellularly (Zhou andVollrath, 1999; Caballero et al., 2000; Caballero and Borras, 2001; O’Brien et al., 2000;Wentz-Hunter et al., 2004). Therefore, it is of importance to identify intracellularbinding partners for myocilin, such as proteins and subcellular structures, that maymediate its action (Wentz-Hunter et al., 2002; Fautsch et al., 2004).

γ-Synuclein is a member of the synuclein family (Buchman et al., 1998a,b)Lavedan et al., 1998; Surguchov et al., 2001a,b; associated with some alterationsin glaucomatous ONH (Surgucheva et al., 2002)). It is a small (14.5 kDa) solu-ble protein that might be associated with centrosomes in some cell types. It isexpressed in the retina and ONH, as well as in other tissues (Buchman et al.,1998a; Fung et al., 2003; Surguchov et al., 2001a,b). Another member of the synu-clein family, α-synuclein, is involved in Parkinson’s disease (PD) and other neu-rodegenerative disorders (Iwai et al., 1995; Polymeropoulos et al., 1997; Claytonand George, 1999; Kruger et al., 1998; Duda et al., 1999). Both mutations in α-synuclein (Polymeropoulos et al., 1997; Kruger et al., 1998) and elevated expres-sion of the wild-type protein (Singleton et al., 2003) may cause the early-onsetform of PD. Synucleins are heat-shock proteins or chaperones and their inter-action with different protein targets may considerably alter protein properties,leading to significant consequences for the cell (Souza et al., 2000; Giasson et al.,2003; Jiang et al., 2004). It is known that in ocular tissues, γ-synuclein is expressedin RGC, ON astrocytes, HTM, and in other cells at a lower level (Surguchovet al., 2001a,b; Farkas et al., 2004). According to recent gene profiling analysis,γ-synuclein is among the largest RGC clusters (Farkas et al., 2004).

In the present study, we searched for γ-synuclein-interacting proteins. Throughimmunoprecipitation using γ-synuclein-specific antibody, we found myocilin amongother interacting proteins. Subsequent experiments revealed that γ-synuclein andmyocilin have partially overlapping intracellular localization. More importantly, in-teractions between γ-synuclein and myocilin affect the properties of the latter, alter-ing its aggregation and secretion pattern. These results suggested that the interactionof these proteins might be involved in glaucoma development.

METHODS

Culture of the Rat Retinal Ganglion Cell Line RGC-5

Culture of RGC-5 cells was kindly provided by Neeraj Agarwal (Krishnamoorthyet al., 2001) (University of Oklahoma Health Sciences Center). Cells were maintainedin Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum


(FBS), 4 mM glutamine (Gibco BRL, Gaithersburg, MD), 100 U/ml penicillin, and100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO). The cells were grown in ahumidified atmosphere of 95% air and 5% CO2 at 37◦C. The RGC-5 cells have adoubling time of approximately 18–20 h and are passaged by trypsinization every3–4 days as previously described (Krishnamoorthy et al., 2001).

Human TM Cells

Fourth- or fifth-passage HTM cells derived from 15- and 22-year-old donorswithout symptoms of ocular diseases were cultured in 25-cm2 tissue culture flasks withEagle’s minimum essential medium including 10% FBS, 5% calf serum,2% essential, and 1% non-essential amino acids, 10 µg/ml gentamicin, and 1.25 µg/mlfungizone (Wentz-Hunter et al., 2004).

HT22 Cell Culture

The mouse HT22 hippocampal cell line was maintained in Dulbecco’s mod-ified Eagle’s medium (Invitrogen, Carlsbad, CA) containing 10% FBS, 100 U/mlpenicillin, and 100 (g/ml streptomycin (Sigma-Aldrich, St. Louis, MO).

Immunofluorescent Staining

For single and double staining, RGC-5 or HTM cells were split on glass cover-slips at ∼50–60% confluency and kept at 37◦C in 5% CO2. The coverslips were pre-treated overnight at 37◦C with laminin (10 µg/ml) and subsequently at room temper-ature (RT) for 30 min with poly-L-lysine (0.1 mg/ml). Cells were washed briefly withphosphate buffered saline (PBS), fixed at −20◦C for 10 min with 100% methanol,and then for 4 min at −20◦C with a methanol:acetone mixture (1:1). After washingseveral times with PBS, the coverslips were blocked by incubation for 1 h at RT withPBS containing 1% BSA, 0.1% of Triton X-100. For staining, the coverslips wereplaced down on a drop of primary Ab (20–40 µl) diluted in blocking solution and leftovernight at 4◦C in a humidified chamber. This procedure was used for γ-synucleinand γ-tubulin staining. Monoclonal anti-γ-tubulin (Sigma-Aldrich, St. Louis, cloneGTU-88) was used in 1:5000 dilution.

For actin and myocilin double staining, cells were fixed at RT for 10 min in4% PA (Sigma-Aldrich, St. Louis, MO) and permeabilized at RT for 30 min in0.1% Triton X-100. After intensive washing, samples were incubated with primaryγ-synuclein antibody at 1:100 dilution (ABCam, Cambridge, MA), washed withPBS, and then incubated in rhodamine-conjugated goat-antirabbit IgG (1:200 di-lution) and phalloidin-Oregon Green (1:50), both from Jackson Immuno ResearchLaboratories, Inc. (West Grove, PA). Antirabbit polyclonal Ab produced against theN-terminal fragment of human myocilin (gift from Dr. D. Stamer) was diluted 1:250.After the final wash, coverslips were mounted in Vectashield (Vector, Burlingame,CA). For some staining experiments for human γ-synuclein, an antibody generatedin our lab (Surguchov et al., 2001a) was used. For nuclear staining DAPI (Sigma)was added to the last washing solution at 1:10,000 dilution.


The specimens were analyzed under a Zeiss Axiomat inverted fluorescent mi-croscope equipped with an Axiocam run by OpenLab (Improvision) software. Theimages were captured with a digital camera Jenoptic (Jena).

Generation of Bicistronic Vector Expressing α- and γ-Synuclein

Bicistronic mammalian expression vector pIRES (Clontech) was used to ex-press α- and/or γ-synuclein in one of two multiple cloning sites (MCS), A or B. Thepresence of a cDNA in the A-site ensures its expression at a high level (according toour data with Western blot analysis, six times higher expression than in untransfectedcells), while its location in the B-site ensures moderate levels of expression (two tothree times compared to untransfected cells) (Surgucheva et al., manuscript in prepa-ration). α-Synuclein cDNA was inserted into the A-site using synthesized primers to5′- and 3′- of α-synuclein cDNA with engineered adaptors on their ends correspond-ing to Nhe1 and Xho1 recognition sequences. To insert α-synuclein into the B-site, theset of primers corresponding to 5′- and 3′-α-synuclein sequences contained Xba1 andNot1 adaptors. Using polymerase chain reaction (PCR), amplification products withthe size of 423 base pairs (bp) for wild-type α-synuclein were generated. γ-SynucleincDNA (384 bp) was inserted in the A-site of the pIRES vector after amplificationwith primers containing engineered restriction sites NheI/EcoRI. For insertion ofγ-synuclein cDNA in the B-site of pIRES, primers with engineered XbaI/NotI siteswere used. DNA sequence analysis confirmed that the sequences of all inserts werecorrect.

Generation of stable clones overexpressing α- and γ-synuclein. The mouseHT22 cell line was grown in DMEM supplemented with 10% FBS and 1% peni-cillin/streptomycin. For each experiment, cells were plated at 1–3 × 105 cells in 2 mlof media in a 35-mm plastic culture dish. Transfection was carried out when cellsreached between 50 and 80% confluency. DNA solution (1.0 µg) was added to predi-luted FuGENE 6 (Roche, Indianapolis, IN). The cells were incubated for 45 min atRT with regular culture medium. Afterwards, the DNA/FuGENE mixture was addeddropwise to the cells and the incubation continued. After 48 h, the cells were dis-sociated by trypsin and replated at a density of 1.0–1.5 × 105 cells/100 mm dish inthe presence of 400 µg/ml of G418 (Gibco, Carlsbad, CA). Stable clones expressingsynucleins were isolated by using the limited dtableilution method. After 3 weeks ofgrowth in selective medium, G418 resistant colonies were cloned.

Western Blotting

For analyses of overexpression, the cells were disrupted using an UltrasonicDismembrator (Fisher Scientific) and the extract was evaluated by Western blotting.Total protein (10–20 µg) was resolved by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). After electrophoresis, proteinswere transferred onto nitrocellulose membrane at 0.45 µM (BioRad, Hercules, CA).Nonspecific binding sites were blocked by immersing the membrane for 1 h at RTin 5% blocking reagent in Tris-buffered saline Tween (TBS-T) on an orbital shaker.The membrane was probed with rabbit polyclonal anti-γ-synuclein (ABCam, 1:1000,


specific for human and rat). For α-synuclein, either monoclonal syn1 (BD-Transduction Lab) or polyclonal AB5038 (Chemicon, Temecula, CA) was used.Four different myocilin Abs were used in this study. For mouse myocilin detectionpolyclonal antirabbit serum against mouse myocilin-GST fusion protein was usedin dilution 1:1000. Myocilin fragment in this fusion protein contained AA 100–187.For the majority of experiments with human myocilin we used antirabbit polyclonalAb against N-fragment of myocilin in dilution 1:2500–1:1500 (gift of Dr. Stammer).For detection of the N-part of human myocilin we used affinity purified antirabbitTIGR-ABI (651) produced against leucine zipper domain (AA 108–131; dilution1:1000, gift of Dr. Fautsch). To detect rat myocilin (in RGC-5 cells) and C-terminalpart of human myocilin we used affinity purified antirabbit TIGR-AB3 (653) pro-duced against olfactomedin domain of myocilin (AA 403–426; dilution 1:500–1:1000,gift of Dr. Fautsch).

A rabbit polyclonal antiserum raised to a synthetic peptide corresponding toAA 595–611 in β-amyloid precursor protein (sAPP) was a gift from Dr. D. Selkoe(Harvard Medical School, Brigham and Women’s Hospital). Serum designatedR1736 was used in dilution 1:2000.

Horseradish peroxidase-conjugated antirabbit IgG (Amersham Pharmacia,Piscataway, NJ) was diluted 1:40,000. Horseradish peroxidase-conjugated antimouse-IgG (Amersham-Pharmacia, Piscataway, NJ) was used in 1:20,000 dilution. The ECLdetection system (Amersham Pharmacia) was used for the detection of the primaryantibody as recommended by the manufacturers. Experiments were repeated threetimes and the intensity of the bands on the autoradiogram was quantified with KodakImage Station 2000R image analyzer using KODAK 1D Image Analysis Software.

Protein Assay

The amount of protein in samples was determined using the BCA Protein AssayReagent (Pierce, Rockford, IL). A two-step approach was used to ensure that weapply an equal amount of proteins in each well. First we measured protein, thenmade a necessary dilution and after this we measured protein once more to be surethat the protein amount became equal after adjustment.

MS/MS Analysis

In-gel proteolytic digests were analyzed by HPLC-nanoelectrospray ionizationtandem mass spectrometry (NESI-MS/MS) on the LCQ Deca XP Plus ion trap massspectrometer (ThermoFinnigan) coupled to turbo-SEQUEST database search withBioWorks 3.0 software (TermoFinnigan). The peptide sequences were identified byrespective tandem mass spectra.

Reverse Transcription (RT)-PCR

Total RNA was extracted from HTM cells using the RNeasy Mini Kit (Qiagen,Valencia, CA). cDNA was prepared employing random hexamers according to themanufacturer’s protocol (SuperScript First-Strand Synthesis System, Gibco BRL).


Amplification of synuclein’s cDNAs was carried out as follows. PCR was conductedusing α-synuclein-specific sense (α-synS: 5′-ATGGATGTATTCATGAAAGG-3′)and antisense (α-synAS: 5′-TGCTGCAATGCTCCCTGCTC-3′) primers andγ-synuclein-specific sense (γ-synS: 5′-ATGGATGTCTTCAAGAAG-3′) and an-tisense (γ-synAS: 5′-GGTCACGCTCTGTACAACATTCTC-3′) primers. The ex-pected sizes of PCR products for α- and γ-synuclein were 271 and 156 bp, respec-tively. Aliquots of cDNA samples were amplified using 0.2 µM of each synucleinprimers and Taq polymerase (Qiagen) on a GeneAmp PCR system 9700 (PE AppliedBiosystem, Norwork, CT). The conditions were: one cycle of 94◦C, 2 min followedby 35 cycles of 94◦C, 30 s; 55◦C, 30 s; and 72◦C, 30 s followed by 10 min incubation at72◦C.

Mouse myocilin cDNA was amplified using total RNA extracted from HT22cells as described above. The oligos specific for mouse myocilin were: S3 (exon 2)5′-GTTGGCCTTCCAGGAATTGA-3′ and AS3 (exon 3) 5′-GTCAATCCTCCATGTGCTTT-3′. The conditions of PCR were identical to that for synuclein amplifi-cation described above except that the annealing temperature was changed to 60◦C.RT-PCR products with the size of 241 bp were cloned into a cloning vector andsequenced.

Co-Transfection of Myocilin and γ-Synuclein into RGC-5 Cells

The cells were split into 12-well plate at a density of 105 cells/ml in regular medialacking antibiotics. They were incubated at 37◦C for 6 h and re-fed with fresh culturemedium prior to transfection. For transfection, 3–6 µl of FuGene 6 (depending on thevolume of DNA) and 50–500 ng of γ-synuclein-pcINeo and myocilin-pCS2-vectorswere used. Human myocilin was amplified using oligos containing adaptor sequencesfor BamH1 (5′- end) and ClaI (3′-end) restriction endonucleases and inserted intopCS2 vector. After 72 h, the medium was withdrawn and the cells were incubated foran additional 48 h in serum-free ITS media (Becton Dickenson, Bedford, MA). Insome samples, ITS medium containing 100 nM dexamethasone (DEX) was added.The medium was subsequently withdrawn and concentrated 10 times by filtrationthrough Amicon PM-30 membrane. The cells were disputed and cell extracts wereobtained as described above.

HT22 Clones Overexpressing γ-Synuclein

Generation of γ-synuclein overexpressing clones is described in Section “Gen-eration of bicistronic vector expressing α- and γ-synuclein.” The clones were grownuntil confluency in 2 mm × 150 mm plates in regular media containing 400 µg/ml ofG418. Cell extracts were obtained as described in Section “Immunoprecipitation.”Cell extract (100 µl) with protein concentration 3.2 mg/ml were pre-cleared using20 µl of normal antigoat or normal antirabbit serum (Calbiochem). After incuba-tion at +4◦C for 30 min 20 µl Protein-G agarose (Sigma-Aldrich, St. Louis, MO)was added and the sample was kept shaking for another 30 min. Twenty micro-liters of goat anti-γ-synuclein (200 µg/ml, Santa Cruz, SC 10698), or 10 µl of normalgoat serum as a negative control were added to pre-cleared extract. The samples


were incubated overnight at +4◦C as described above. Antirabbit myocilin serumwas used for Western blot analysis in dilution 1:1000 for detection of protein afterimmunoprecipitation.

Immunoprecipitation

HTM cells grown in two 25 cm2 flasks were washed by cold PBS and sus-pended into 100 µl of IP buffer (50 mM Tris–HCl, pH 7.4, 5 mM EDTA, 0.25 M NaCl,0.5% Nonidet P-40), containing protease inhibitor cocktail. The cells were sonicatedand the cell debris was removed by spinning at 12,000 rpm for 15 min. Pre-clearedsupernatant was incubated overnight with 20 µl of goat anti-γ-synuclein antibody(Santa Cruz, CA, SC-10698, concentration 200 µg/ml). The sample was further in-cubated for 2 h at 4◦C with 20 µl of Protein G suspension (Sigma). The immuno-precipitate was collected by 10 min centrifugation at 12,000 rpm and washed threetimes in IP buffer. Finally 2× sample buffer was added to the supernatant and beadspellet and boiled for 3 min. The samples were loaded onto 12% polyacrylamide gels,electrophoresed, transferred to PVDF- or NC-membrane, and probed with rabbitanti-γ-synuclein antibody (ABCam) (dilution 1:1000).

Pull-Down Experiments

cDNA fragments corresponding to N- and C-terminal domains of human my-ocilin were inserted into BamH1–EcoRI restriction sites of bacterial expressionvector pGEX-2T (Amersham Pharmacia) to produce GST fusion proteins. Recom-binant protein expression in E. coli BL21 was induced by adding 0.5 mM IPTG(Promega, Madison, WI) and incubating at 27◦C for 3 h. GST fusion proteins werepurified on glutathione-agarose beads. γ-Synuclein cDNA was cloned into bacte-rial pTRcHisA vector (Invitrogen, Carlsbad, CA) and expressed in BL21 cells afterIPTG induction. Cell extract was loaded on ProBond column and recombinantγ-synuclein was eluted by 20 mM Na–phosphate buffer, pH 6.0, 500 mM NaCl, and300 mM imidazole. Recombinant γ-synuclein was dialyzed for 6 h at 4◦C againstbinding buffer, containing 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP-40,0.5% NaDOC, 10 mM DTT, and a protease inhibitors cocktail (Roche, Indianapolis,IN). N- and C-fragments of myocilin-GST bound to agarose glutathione beads wasincubated overnight with recombinant γ-synuclein in the binding buffer. After inten-sive washing, bound proteins were eluted with 2× sample buffer containing 20 mMDTT, boiled, and analyzed by 12% SDS–PAGE.

Overlay Experiment

C- and N-myocilin were expressed in E. coli using pGEX2T vector, puri-fied on glutathione-agarose beads as it is described for pull-down experimentsabove and treated with thrombin to eliminate GST. For this purpose myocilinfragments were incubated in STE buffer (10 mM Tris–HCl, pH 8.0, 100 mM NaCl,and 1.0 mM EDTA) for 1 h at 30◦C. After incubation the beads were thoroughlywashed and myocilin fragments were eluted from agarose beads by 2× SDS buffer.


Myocilin fragments were separated in 12% PAGE, blotted on PVDF with subsequentincubation of the blot with recombinant γ-synuclein (10 µg/ml) or binding buffer(100 mM KCl, 50 mM Tris—HCl, pH 7.4, 1.0 mM EGTA, 2 mM MgCl2, 1 mM DTT,and 0.2% Tween 20) at 4◦C overnight. Before overlay assay recombinant γ-synucleinwas dialyzed against the buffer for 6 h at 4◦C. After incubation with γ-synucleinthe blot was washed three times with binding buffer, probed with γ-synuclein Ab(ABCam, dilution 1:1000) for 2 h at RT and then incubated with HRP-antirabbitconjugated (dilution 1:40,000).

RESULTS

Co-IP of Myocilin and γ-Synuclein

Initially we detected myocilin among γ-synuclein-binding proteins when weused IP of HT22 proteins by γ-synuclein-specific Ab. For this purpose, we usedstable clones of HT22 overexpressing γ-synuclein. This culture of hippocampal neu-rons produces sufficient amounts of endogenous myocilin for studies of myocilin–γ-synuclein interactions without myocilin overexpression.

IP of HT22 cell extracts with anti-γ-synuclein followed by immunoblotting re-vealed the presence of myocilin in the immunoprecipitated pellets (Fig. 1A). Myocilinwas observed as a 52 kDa protein in immunoprecipitates when IP was performedwith goat anti-γ-synuclein antibody (Fig. 1A, lanes 1–3). Similar band was detectedwhen we used rabbit instead of goat anti-γ-synuclein antibody (not shown). Thisband was practically absent when normal goat serum instead of anti-γ-synuclein wasused as a control (lane 4).

To identify a myocilin fragment responsible for binding to γ-synuclein we pu-rified recombinant C- and N-myocilin fragments, separated them in 12% PAGE,blotted on PVDF, and incubated with recombinant γ-synuclein (Fig. 1B, lanes 1and 2) or binding buffer (Fig. 1B, lanes 3 and 4). γ-Synuclein was detected as aprotein bound to C-fragment of myocilin (lane 1), but not N-fragment (lane 2). Thisband was further characterized by HPLC-NESI-MS/MS analysis of the in-gel prote-olytic digest coupled to turbo-SEQUEST database search. Myocilin was identifiedwith the significant score 10.2, based primarily on the high-resolution tandem massspectrum of the peptide IDTVGTDVRQ from C-fragment of human myocilin.

Interaction between myocilin and γ-synuclein was further confirmed by pull-down experiments. Pull-down assays using GST-human myocilin immobilized onagarose-glutathione beads showed that γ-synuclein was present in the bound proteinseluted with 2× sample buffer and 20 mM DTT from the beads. It was identified asa protein with molecular weight ∼17–18 kDa immunoreactive to anti-γ-synucleinantibody (data not shown). No corresponding protein band was noted when GSTwithout myocilin was used for immobilization.

γ-Synuclein and Myocilin Expression and Localization in Cultured Cells

The finding that myocilin was one of the γ-synuclein-binding proteins inbiochemical experiments prompted us to determine their expression patterns and


Fig. 1. Myocilin–γ-synuclein interaction. (A) Co-IP of myocilin with γ-synuclein. Extractsof HT22 cells were preincubated overnight with normal goat serum. To reduce nonspecificbinding, the extracts were treated with Protein G. IP was performed with pre-cleared super-natants using goat anti-γ-synuclein (lanes 1 and 2), or normal goat serum (lanes 3 and 4).Immunoprecipitates were analyzed by Western blotting with mouse antirabbit myocilin.Lanes 1 and 3: Supernatants not bound to Protein G; lanes 2 and 4: Immunoprecipitatesbound with protein G and eluted by sample buffer. Arrow to the right shows myocilin band.(B) Detection of γ-synuclein binding to recombinant myocilin fragments. Purified recom-binant C- and N-myocilin fragments were separated in 12% PAGE, blotted on PVDF, andincubated with recombinant γ-synuclein (lanes 1 and 2) or binding buffer (lanes 3 and 4).γ-Synuclein was detected in a band corresponding to C-fragment of myocilin (lane 1, arrow),but not N-fragment (lane 2).

intracellular localization in the two cell types routinely used to study molecularand cellular mechanisms of glaucoma, i.e., immortalized rat ganglion cells (RGC-5)and human trabecular meshwork cells. The results obtained using these two celllines of ocular origin were compared with those from the myocilin-expressing HT22neuronal cultures.

RGC-5 Cells

The expression of γ-synuclein in ganglion cells has been demonstrated earlierby immunohistochemical staining of human retina (Surguchov et al., 2001a). Cur-rent analyses by Western blotting further confirmed the presence of a monomericform (17 kDa) and aggregated forms (molecular weights of ∼35 and 68 kDa; pre-sumably, dimer, and tetramer) (Fig. 2A, lane 1) of γ-synuclein in RGC-5 extracts.The aggregates disappeared after one cycle of freezing and thawing (Fig. 2A, lane 2).

Both the endogenous rat (Fig. 2B, lane 5) and the human recombinant (Fig. 2B,lane 6) myocilin could be identified in RGC-5 extracts as a protein with


Fig. 2. Detection of synucleins and myocilin in HTM and RGC-5 cells.(A) Detection of γ-synuclein in RGC-5 cells. The cell extracts were preparedas described in Section “Methods”. Total protein (40 µg) was loaded on 12%PAAG and γ-synuclein was detected by Western blot analysis using rabbitanti-γ-synuclein antibody. Lane 1: Freshly prepared sample; lane 2: The sam-ple after freeze and thaw cycle; lane 3: “Rec” shows position of the fusionrecombinant γ-synuclein with His-tag used as a positive control. (B) Detec-tion of myocilin in RGC-5 cells. Equal amount of the total protein from con-trol RGC-5 cells (lanes 1, 3, and 5) and cells overexpressing human myocilin(lanes 2, 4, and 6) were resolved on 10% polyacrylamide gel and immuno-probed with polyclonal rabbit antibody against the N-terminal fragment ofmyocilin (gift of Dr. D. Stamer) (lanes 1 and 2), TIGR-AB1#651(108–131AA)(lanes 3 and 4), or TIGR-AB 3#653 (403–426 AA) (lanes 5 and 6). (C) De-tection of α- and γ-synucleins in HTM cells. Lanes 1 and 2: Cell extractswere immunoprecipitated by goat anti-γ-synuclein antibody and probed withrabbit anti-γ-synuclein (1:1000). Lane 1: Supernatant after immunoprecipi-tation. Lane 2: Pellet after immunoprecipitation. Lanes 3 and 4: The sameblot reprobed with α-synuclein antibody syn1 (1:750). Arrow 1, position ofheavy chains of IgG; arrow 2, light chain of IgG; arrow 3, α-synuclein; arrow4, γ-synuclein. (D) Identification of α- and γ-synuclein messages by RT-PCR.PCR was performed using cDNAs synthesized from total RNA of HTMcells (lanes 2 and 4) or control vectors containing synuclein cDNAs (lanes 3and 5). PCR amplification was carried out with α-synuclein primers (α-syn-Sand α-syn-AS) and cDNA from TM cells (lane 2); α-synuclein primers andα-synuclein cDNA in vector pCEP4 as a control (lane 3); γ-synuclein primers(γ-syn-S and γ-syn-AS) and cDNA from TM cells (lane 4); and γ-synucleinprimers and γ-synuclein cDNA in vector pCINeo as a control (lane 5). Mark-ers VI (Roche, Indianapolis, IN) are shown in lanes 1 and 6. The sizes of the11 markers from the top are 2176, 1769, 1230, 1033, 653, 517, 453, 394, 298,234, and 164 bp, respectively. Arrow 1, position of α-synuclein PCR product(271 bp); arrow 2, γ-synuclein product (156 bp).

molecular weight 55 kDa by an antibody raised against the olfactomedin-like portion(AA 403–426) of the protein (#653-TIGR-AB3). At the same time, antibodiesspecific to the leucine-zipper domain (#651-TIGR-AB1;108–131 AA) and the N-fragment of human myocilin recognized only the human recombinant myocilin(Fig. 2B, lanes 2 and 4), but not the endogenous rat myocilin (Fig. 2B, lanes 1


Fig. 3. Immunofluorescence staining of RGC-5 cells with anti-γ-synuclein and anti-γ-tubulin antibodies(A–C) and γ-synuclein/myocilin (D–F). (A–C) Non-transfected cells. (D–F) Cells transfected to overex-press γ-synuclein and myocilin as described in Section “Methods”. (A) shows the staining for γ-synuclein(green) and (B) shows the γ-tubulin (red) staining in dense RGC-5 cultures. The merged image is shownin (C). (D) shows the staining with rabbit anti-human myocilin (#651, 1:250) (green) and (E) shows thestaining with goat anti-γ-synuclein (1:50) (red) in cells grown at low density. (F) is the merged imageof (D) and (E). Antirabbit Oregon green (1:100) and donkey antigoat IgG conjugated with rhodamineRed (Santa Cruz) was used for γ-synuclein detection. Arrows show centrosomal staining. Experimentsrepeated a minimum of four times each and a representative cell is shown. Bar :10 µm.

and 3). The lack of immunoreactivity of the N-terminal antibodies with rat proteinis anticipated, since the amino acid sequence at the N-terminus of human myocilinis different from that of the rat ortholog. The C-terminus sequence of myocilin fromthe two species is highly homologous.

Immunofluorescence staining of RGC-5 cells with anti-γ-synuclein demon-strated a diffuse intracellular localization with a higher level of staining displayed inperinuclear areas (Fig. 3A and C). In some cells, staining of round subcellular struc-tures was partially overlapping (Fig. 3C, arrows) with that of γ-tubulin, a centrosomemarker (Fig. 3B, arrows). Co-transfection of γ-synuclein and myocilin did not alterγ-synuclein localization in RGC-5 cells (Fig. 3E and F). The γ-synuclein localiza-tion also remained unchanged even when RGC-5 cells altered shape at differentcell densities (compare Fig. 3A–C and D–F). Partial co-localization of γ-synucleinwith myocilin in perinuclear area can be seen in a broad area on merged images(Fig. 3F, yellow, arrows). A similar cytoplasmic/perinuclear myocilin localization(Mertts et al., 1999; O’Brien et al., 2000; Wentz-Hunter et al., 2003) and its putativeassociation with centrosomes (O’Brien et al., 2000; Ueda et al., 2000; Noda et al.,2000) have been described previously in some cell types.

HTM Cells

The expression of both γ-synuclein and α-synuclein in HTM cells was evidencedby Western blotting (Fig. 2C, lanes 2 and 3). α-Synuclein could be detected in HTM


Fig. 4. Staining of HTM cells. Human TM cells were plated on laminin or poly-L-lysine-treated cover-slips. Two days later, the cells were fixed and stained for actin (A, green), endogenous γ-synuclein (B,red), actin and γ-synuclein (C, merged image), or endogenous myocilin (D, green). Nuclei were stainedby DAPI in blue. Bar :10 µm. (E) shows the same image as in (D) but stained with DAPI. (F) is themerged image of (D) and (E). Experiments repeated a minimum of four times each and a representativecell is shown. Arrows show centrosomal staining.

extracts directly, while the presence of γ-synuclein as a protein with molecular weight∼17–18 kDa could be reliably shown only after IP (Fig. 2C, lane 2, arrow 4).

The expression of α- and γ-synucleins in HTM cells was further confirmed byidentification of their messages using RT-PCR analysis. Amplification of cDNAssynthesized from total RNA of HTM cells with primers to α-synuclein (Fig. 2D, lane2) and γ-synuclein (Fig. 2D, lane 4) produced PCR products with the anticipatedsizes (271 and 156 bp, respectively), corresponding to the sizes of products gener-ated on control templates (cDNAs for α- and γ-synuclein in vectors pCEP4 andpcINeo, Fig. 2D, lanes 3 and 5, respectively). By PCR, γ-synuclein message couldbe identified only after 35 cycles of amplification (Fig. 2D, lane 4), indicating that itslevel of expression in HTM cells is relatively low. In contrast to the published data(Tanji et al., 2002), pretreatment of HTM cells with IL-1β did not affect the level ofγ-synuclein expression in this cell type (not shown).

By immunofluorescence, γ-synuclein has a diffused cytoplasmic localization inHTM cells (Fig. 4B and C, red staining). Myocilin has a similar diffused localizationand often is enriched in perinuclear area (Fig. 4D and F, green staining).

Preliminary experiments showed that myocilin staining was distributed evenlyin HTM cells from healthy individuals, whereas only some cells were stained verybrightly and others did not show staining in samples from POAG. Similarresults were obtained previously by Stammer and coauthors (personalcommunication).

Surprisingly, myocilin expressed in another type of neuronal culture–HT22could be detected by Western blotting analysis without preliminary immunopre-cipitation (see Section “Effect of γ-synuclein on myocilin secretion in HT22 cells”


later). At the same time, we did not find myocilin in other neuronal cultures, e.g.,mouse dopaminergic neurons MN9D and motor neurons NSC34. To confirm the ex-pression of mouse myocilin in hippocampal neuron culture we isolated RNA, usedRT-PCR and sequenced PCR products generated with primers specific to exon 2(sense primer) and exon 3 (antisense primer). The sequence of the PCR product was100% identical to mouse myocilin sequence.

Effect of γ-Synuclein on Myocilin Secretion in HT22 Cells

Western blot analyses and quantitation by densitometry indicated that the rela-tive amount of myocilin in the culture media (M) was 2.2-fold lower (Fig. 5A, lane 5)from HT22 cells overexpressing γ-synuclein than that of controls (Fig. 5A, lane 1) orcells overexpressing α-synuclein (Fig. 5A, lane 3). The reduction of secreted myocilinwas also observed in culture media of clones overexpressing γ-synuclein togetherwith α-synuclein (Fig. 5A, lanes 7 and 9).

At the same time, intracellular myocilin level was even slightly higher in clonesoverexpressing γ-synuclein (Fig. 5A, lanes 6 and 10) compared to control cells(Fig. 5A, lane 2). This suggested that myocilin secretion was reduced in clones over-expressing γ-synuclein. By contrast, α-synuclein did not inhibit myocilin secretion,since the amount of secreted myocilin in culture media of α-synuclein overexpress-ing clones was 1.8-fold higher (Fig. 5A, lane 3) than in controls (lane 1). Therefore,γ-synuclein, but not α-synuclein, inhibited secretion of myocilin from cells to culturedmedia. Secretion of other proteins such as secreted form of β-amyloid precursor pro-tein (sAPP) was not altered in the media from γ-synuclein overexpressing cells (notshown) suggesting that the effect of γ-synuclein may be specific for myocilin.

In the presence of 100 nM DEX, the level of secreted myocilin was 4.6 timeshigher (compare lanes 1 in Fig. 5A and B), while intracellular level of myocilinwas slightly reduced (Fig. 5A and B, lanes 2) in HT22 cells. DEX thus appeared tostimulate myocilin expression and secretion in HT22 cells as in other cell types (Clarket al., 2001; Tamm, 2002). To our knowledge, myocilin upregulation by DEX has notbeen described before for neuronal cultures. Furthermore, the inhibiting effect ofγ-synuclein on myocilin secretion persisted in the presence of DEX (compare lanes1 and 9 in Fig. 5B). The inhibition of myocilin secretion was not as pronounced asthat observed in the absence of DEX (Fig. 5A). It is possible that with the elevatedamount of myocilin induced by DEX, the concentration of γ-synuclein may not besufficient for inhibition.

Effect of γ-Synuclein on Myocilin Aggregation in HT22 Cells

As can be seen further from Fig. 5A, HT22 cells expressed myocilin both as amonomer with molecular weight of 52 kDa and aggregates with molecular weight inthe 120–200 kDa range (Fig. 5A, lanes 2 and 4). In cells overexpressing γ-synucleineither alone or together with α-synuclein, the amount of myocilin aggregates wasconsiderably reduced (lanes 6, 8, and 10). This γ-synuclein effect may be specific,since another member of the synuclein family, α-synuclein, did not inhibit myocilinaggregation (Fig. 5A, lane 4).


Fig. 5. Effect of α- and γ-synucleins on secretion andaggregation of myocilin. (A) Intracellular and secretedmyocilin without DEX. Stable clones overexpressing γ-synuclein were generated as described in Section “Meth-ods.” The cells were split into 100 mm2 cell dishes andgrown for 48 h. After the regular media was changedfor serum-free media the cells were grown addition-ally for 72 h. Then, the media was collected and con-centrated 10 times using Amicon PM-10 device. Totalprotein (4 µg) was loaded on each lane of 10% poly-acrylamide gels and immunoprobed with rabbit anti-myocilin antibody, generated against mouse antigen.Both HT22 cells lysates (C, lanes 2, 4, 6, 8, and 10)and the corresponding cultured media (M) (lanes 1, 3,5, 7, and 9) were analyzed. Lanes 1 and 2: Controls(cells transfected by an empty vector); lanes 3 and 4:Cells overexpressing α-synuclein; lanes 5 and 6: Cellsoverexpressing γ-synuclein; lanes 7 and 8: Cells trans-fected to express a high concentration of α-synucleinand a low concentration of γ-synuclein; lanes 9 and10: Cells transfected to express a high concentration ofγ-synuclein and a low concentration of α-synuclein. Ex-periment was performed in triplicate. (B) Intracellularand secreted myocilin after DEX treatment. HT22 cellswere treated with dexamethasone. The cells were platedonto 2 mm × 35 mm plates at regular density for 2 days,and then the media was changed into serum-free con-ditions. To one of the plates, DEX was added in a finalconcentration of 100 nM. After 72 h of growth the cellswere harvested and the conditioned medium was con-centrated. Protein (5 µg) was loaded on the 10% poly-acrylamide gels and probed as described in the legendto Fig. 5A. Lane designation is identical to Fig. 5A.

Effect of γ-Synuclein on Myocilin Expression and Secretion in RGC-5

To further confirm the effect of γ-synuclein on myocilin expression and secre-tion, we co-transfected RGC-5 cells with increasing amount of expression vectorscontaining γ-synuclein and myocilin. As shown on panel E of Fig. 6, the level of


Fig. 6. Effect of various doses of γ-synuclein on my-ocilin expression and secretion in RGC-5 cells. (A) and(B) RGC-5 cells were transfected with 50 ng (lanes 1 and4), 100 ng (lanes 2 and 5), and 250 ng (lanes 3 and 6) my-ocilin cDNA (MyoC) in pCS+. Forty-eight hours aftertransfection, the cells in (A) were incubated with serum-free medium. Cells in (B) were in addition treated with100 nM DEX for 4 days. The cell extracts (lanes 1–3)and culture medium (lanes 4–6) were separated by 10%SDS–PAGE and probed by polyclonal human antirabbitmyocilin (dilution 1:2,800). Arrow 2, position of intra-cellular non-processed myocilin; arrow 3, maturated se-creted myocilin; arrow 1, diffuse nonspecific staining dueto cross reaction with albumin. (C) and (D) RGC-5 cellswere transfected with 50 ng myocilin + 50 ng γ-synucleincDNA in PCIneo (lanes 1 and 4), 100 ng myocilin + 100 ngγ-synuclein cDNA (lanes 2 and 5), or 250 ng myocilin +250 ng γ-synuclein (lanes 3 and 6). Forty-eight hours aftertransfection, the cells in (C) were incubated with serum-free medium. Cells in (D) were in addition treated with100 nM DEX for 4 days. The cell extracts (lanes 1–3) andculture medium (lanes 4–6) were separated by 10% SDS–PAGE and probed by polyclonal human antirabbit my-ocilin (dilution 1:2800). Experiment was performed in trip-licate. (E) RGC-5 cells were with 50, 100, or 250 ng (lanes2–4, respectively) of γ-synuclein cDNA in pcNeo vector.Lane 1, extracts from non-transfected cells. Samples iden-tical to those applied on lanes 1–3 (panel C) were loadedon 12% PAAG and probed with polyclonal rabbit anti-γ-synuclein antibody (dilution 1:1000).


Fig. 7. Effect of γ-synuclein on truncated Gln368Stopmyocilin secretion in RGC-5 cells. Cells were transfectedwith 500 ng of pCS2-368Stop mutant alone (lanes 1 and 2),or co-transfected with 250 ng of pCS2-Gln368AStop +250 ng pCI-Neo-γ-synuclein (lanes 3 and 4) or with250 ng of pCS2 –Gln368Stop + 250 ng β-synuclein-pCEP4(lanes 5 and 6). Transfection was carried with TM PlusReagent kit. Subsequently, the cells (lanes 1, 3, and 5)and conditioned medium (lanes 2, 4, and 6) were collectedfor immunoblot analyses with anti-myocilin antibody #651(1:1000).

γ-synuclein expression increases progressively with the increasing amounts of ex-pression vector pcNeo-γ-synuclein used for transfection.

As one can see in Fig. 6A, higher expression of myocilin in RGC-5 cells isobserved with increasing amount of expression vector pcNeo-γ-synuclein (panelA, lanes 1–3). Significant myocilin secretion occurred when we used 250 ng DNA(panel A, lane 6). In cells overexpressing γ-synuclein, the level of myocilin expressionwas 3.8 times higher compared to cells expressing myocilin alone (Fig. 6, comparelanes 1, 2, and 3 on panels A and C). The highest amount of secreted myocilinwas found at 250 ng DNA after the cells were treated for 4 days with 100 nM DEX.When the cells were co-transfected with myocilin and γ-synuclein expression vectors(panel C, lanes 1–3), we detected only intracellular, but not secreted myocilin (panelC, lanes 4–6). In the presence of 100 nM DEX the inhibition of myocilin secretionwas observed only at minimal amount of DNA used (panel D, lanes 1 and 4).

To determine the effect of γ-synuclein on secretion of myocilin carrying dif-ferent mutations, wild-type or mutant myocilin (Tyr437His, Ile477Asn, and trun-cated Gln368Stop) was co-transfected with γ-synuclein into RGC-5 cells. Wild-typemyocilin was detected mainly in the conditioned medium, whereas Tyr437His andIle477Asn mutant forms of the protein were present exclusively in the cells (datanot shown). The truncated Gln368Stop myocilin was found both inside the cells and


in culture media. γ-Synuclein slightly reduced the level of truncated Gln368Stopsecretion (Fig. 7, lanes 3 and 4). At the same time, β-synuclein had little effect onthe Gln368Stop secretion.

DISCUSSION

The discovery that mutations in myocilin gene are responsible for some formsof glaucoma (Stone et al., 1997) has had a tremendous impact on glaucoma research.At present, after about 7 years of research on myocilin, more than 50 mutations havebeen identified (Adam et al., 1997; Stone et al., 1997; Allingham et al., 1998; Alwardet al., 1998; Fingert et al., 1999; Michels-Rautenstrauss et al., 1998, 2002; Tamm,2002), and considerable progress has been made in understanding the structure andlocalization of myocilin, as well as the possible mechanisms that control its expression(for review see Tamm, 2002). Nevertheless, the specific function of myocilin in normaleye and its role in glaucoma pathogenesis remain elusive. One of the approaches totry to understand myocilin function is to identify its interacting protein partners.

This study was initiated after finding myocilin among γ-synuclein-interactingproteins by immunoprecipitation (Fig. 1A). Since myocilin mutations are linked tosome forms of glaucoma and γ-synuclein belongs to a family of proteins involvedin several neurodegenerative diseases, it was challenging to investigate whethertheir interaction may occur in vivo and contribute to glaucoma development. Theimportance of this study is strengthened by the fact that members of the synucleinfamily are naturally unfolded and easily bind to other proteins, changing their confor-mation and inducing different pathological conditions (Iwai et al., 1995; Clayton andGeorge, 1999; Souza et al., 2000; Giasson et al., 2003; Snyder et al., 2003; Linderssonet al., 2004). Although functional role of myocilin and synucleins remains elusive,they presumably may be involved in similar processes, i.e., vesicular transport, mod-ulation of extracellular matrix, and regulation of cytoskeleton structure (Claytonand George, 1999; Tamm, 2002; Wentz-Hunter et al., 2002). Interaction betweenmyocilin and γ-synuclein was further confirmed by pull-down experiments and over-lay assay with subsequent MS/MS identification of myocilin C-fragment involved inγ-synuclein binding (Fig. 1B).

In order to study a possible role of myocilin–synuclein interactions we firstdetermined the localization of these proteins in cell types implicated in glaucomapathogenesis.

The results of Western blotting and immunofluorescent staining show thatγ-synuclein is expressed in RGC-5 and HTM cells (Figs. 2–4). It has diffused cy-toplasmic/perinuclear localization. According to published and our experimentaldata (Figs. 2–4), myocilin is also expressed in these cell types and has a similar intra-cellular localization. As was shown earlier, myocilin staining is typically perinuclearin control cells, but with most of the microtubules disassembled by nocodazol, my-ocilin becomes dispersed into numerous punctate bodies or vesicles throughout thecytoplasm (Noda et al., 2000; O’Brien et al., 2000; Wentz-Hunter et al., 2003). Forboth proteins vesicular localization and co-localization with cytoskeleton proteinshave been proposed (Buchman et al., 1998a,b; Clayton and George, 1999; Ueda et al.,


2000). Such intracellular localization of myocilin in HTM cells is in agreement withpublished data for different cell types (Mertts et al., 1999; Noda et al., 2000; O’Brienet al., 2000; Jacobson et al., 2001; Tamm, 2002; Wentz-Hunter et al., 2003, 2004; Fautschet al., 2004). Preliminary experiments showed that myocilin staining was distributedevenly in HTM cells from healthy individuals, whereas only some cells were stainedvery brightly and others did not show staining in samples from POAG. Similar resultswere obtained previously by Stammer and coauthors (personal communication).

Thus, our results presented here and published earlier (Surguchov et al., 2001a,b;Surgucheva et al., 2002) as well as the data by other investigators demonstrate thatboth myocilin and γ-synuclein are expressed in RGC and HTM cells, and have par-tially overlapping intracellular localization. Both proteins are prone to aggregate andhave a similar pattern of expression and localization in cells involved in glaucomapathogenesis. These similarities, as well as their implication in glaucomatous alter-ations and putative participation in similar cellular processes (vesicular transport,modulation of cytoskeleton, possible association with the centrosome, etc.), suggesta high probability of their interaction in vivo.

Since myocilin and γ-synuclein have similar intracellular localization, it is tempt-ing to hypothesize that they interact with each other in vivo. Then an importantquestion arises: If these proteins interact in vivo, what might be the result of such in-teractions? We found several effects of γ-synuclein on myocilin properties. First, thelevel of myocilin secretion and aggregation is significantly changed in the presenceof γ-synuclein (Fig. 5).

The disappearance of myocilin aggregates in the presence of γ-synuclein maybe due to the chaperonic activity of γ-synuclein (Souza et al., 2000; Jiang et al., 2004).Interestingly, the effect of γ-synuclein on myocilin secretion mimics the effect of somemutant forms of myocilin associated with POAG. In several recent publications itwas found that the presence of the mutated myocilin suppresses secretion of thenormal protein (Caballero et al., 2000; Jacobson et al., 2001; Gobeil et al., 2004).

Several putative regions important for dimer and multimer formation wereidentified in myocilin. It has been suggested that a conserved cysteine residue at po-sition 433 in the olfactomedin domain of myocilin contributes to dimerization and/oraggregation (Nguyen et al., 1998). However, recent results suggested that myocilin–myocilin interactions occur mainly in the N-terminal part of protein, containinga leucine zipper domain (AA 117–166) and a coiled-coil motif between residues78 and 105 (Nguyen et al., 1998; Fautsch and Johnson, 2001; Gobeil et al., 2004).Presumably these myocilin regions are also involved in the interaction with otherproteins (Gobeil et al., 2004). Importantly, the involvement of nine cysteine residuesdistributed along the myocilin polypeptide chain can be excluded as candidates forintermolecular disulfide bonding with γ-synuclein, since synucleins have no cysteinsin their sequences. In vitro mutagenesis of amino acids in these regions of myocilinmight give a more precise localization of amino acids responsible for interaction.

Another effect that γ-synuclein exerts on myocilin is upregulation of its ex-pression (Fig. 6A and C). A putative mechanism of this upregulation may involvetranscription factor NF-kappa B and NF-kappa B-inducing kinase (NIK), since ourdata with gene microarray analysis show that γ-synuclein increases 3.1 times thelevel of message for NF-kappa B-inducing kinase (Surguchov et al., manuscript in


preparation). NF-kappa B-inducing kinase is an essential component of NF-kappaB activation (Malinin et al., 1997; Ling et al., 1998), and NF-kappa B plays a rolein regulation of myocilin expression (Kirstein et al., 2000). Therefore, summarizingthe effects of γ-synuclein on myocilin, we found that in cultured cells γ-synucleinupregulates myocilin expression, inhibits its secretion and prevents the formation ofhigher molecular forms of myocilin. However, in intact eye the ratio between theseproteins might be different and therefore additional experiments are necessary tobetter characterize their interaction in ocular tissues.

In the presence of 100 nM DEX the inhibition of myocilin secretion was ob-served only at minimal amount of DNA used (Fig. 6, panel D, lanes 1 and 4). Theseresults might be explained by the fact that γ-synuclein inhibition of myocilin secretionoccurs only at low or moderate myocilin concentration. When myocilin is overex-pressed in the presence of DEX, the amount of γ-synuclein may be not sufficient tocause considerable inhibition.

Previous data on specificity of myocilin and γ-synuclein expression demon-strated that they are expressed in various organs and tissues, including the brain(Stamer et al., 1998; Buchman et al., 1998a,b; Swiderski et al., 1999; Kulkarni et al.,2000; Noda et al., 2000; Jurynec et al., 2003; Ohlmann et al., 2003; Wentz-Hunteret al., 2004). However, recent data concerning cell specificity suggest that they areexpressed predominantly in glial, but not in neuronal cells. It is known that my-ocilin is expressed in CNS glial scar, gliotic tissue, and reactive astrocytes and itsexpression inhibits neurite outgrowth (Jurynec et al., 2003). It is also a componentof the myelin sheath in peripheral nerves and schwannoma cells, however, no orminor expression of myocilin mRNA was found in the brain, spinal cord, and ON(Ohlmann et al., 2003). Our data demonstrating myocilin expression in hipocampalneuron culture shows that myocilin expression may occur in neuronal cells. Becauseof the controversial published data concerning myocilin presence in neurons weconfirmed myocilin expression in neuronal cell cultures by several different meth-ods (ICC, Western blotting, RT-PCR with subsequent sequencing). These resultsdemonstrate that myocilin is expressed in neurons and therefore its putative contri-bution to neuronal regenerative failure characteristic of the mammalian CNS needsfurther investigation.

It is tempting to speculate that myocilin secretion is sensitive to subtle con-formational changes induced by mutations and interaction with different ligands,including other proteins. γ-Synuclein belongs to a class of heat-shock proteins orchaperones that play an important role in controlling protein folding, stabilization oftheir conformation, overall maturation of proteins, and modulation of proteasomalactivity (Clayton and George, 1999; Snyder et al., 2003, 2004; Lindersson et al., 2004).The results presented in this paper demonstrate that myocilin’s interaction withγ-synuclein changes myocilin properties that may have an important role in glau-coma. One of the pathways where their interaction may occur is a secretory pathway,since myocilin is a secreted protein and synucleins are implicated in the modulationof secretory processes (Park et al., 2002; Tompkins et al., 2003).

According to published data, two members of the synuclein family, α- andγ-synuclein have different localization and different pattern of expression. While thelevel of α-synuclein expression is high in neurons, significant γ-synuclein expression


is found in glial cells (Surgucheva et al., 2002; Brenz Verca et al., 2003). α-Synucleinis predominately expressed in the brain and its immunoreactivity is enriched atpresynaptic terminals (Nakaya, 1994; Iwai et al., 1995). At the same time, high level ofγ-synuclein expression is detected in several tumors, and in the olfactory epithelium(Ji et al., 1997; Lavedan et al., 1998; Buchman et al., 1998a,b; Duda et al., 1999).Differential expression of α- and γ-synuclein may be related to functional differencesof the two proteins. Antagonistic effect of different synucleins was recently describedin regulation of proteasomal activity (Snyder et al., in press). Their role as chaperonesmay vary from classical function in helping proteins to acquire correct conformation(Souza et al., 2000) to the role of “pathological chaperone” inducing other proteinfibrillization (Giasson et al., 2003). Here we report that both α- and γ-synuclein areexpressed in HTM cells (Fig. 4); however, only γ-synuclein interacts with myocilinand alters its properties (Figs. 5 and 7). The presence of both synuclein isoforms inHTM cells may reflect the dual nature of this cell type. Although HTM is composedof non-neural cells, they originate embryologically from the neural crest and expressneuron-specific markers, e.g., neuron-specific enolase (NSE), neurotrophins, andneurotrophin receptors (Royds et al., 1982; Tripathi and Tripathi, 1989; Wordingeret al., 2000).

Mutant proteins that fail to fold correctly are often retained within the endoplas-mic reticulum (ER) compartment (Sidrauski et al., 1998; Bross et al., 1999). Severalgenetic and biochemical lines of evidence have suggested that autosomal dominantPOAG-linked myocilin mutations may act through a pathologic gain-of-functionmechanism caused by the intracellular accumulation of mutant proteins (Jacobsonet al., 2001; Caballero et al., 2000; Tamm, 2002; Gobeil et al., 2004). In agreementwith this model, Caballero and Borras presented evidence that a truncated formof myocilin (AA 1–344) was not processed correctly in the ER and accumulatedin insoluble aggregates (Caballero and Borras, 2001). Our data demonstrating thatinteraction of truncated myocilin with γ-synuclein inhibits its secretion suggest thatγ-synuclein might play a role of “pathological chaperone” described earlier foranother member of the synuclein family, α-synuclein (Giasson et al., 2003).

Interestingly, dexamethasone stimulates expression of both endogenous my-ocilin (Fig. 5) and myocilin in expression vector, which lacks myocilin gene upstreamregulatory elements (Fig. 6). This data suggest dexamethasone induction that weobserve might be due to an indirect effect via secondary glucocorticoid-activatedtranscription factors on myocilin expression described earlier (Shepard et al., 2001).

In conclusion, the results presented herein suggest that interaction of myocilinwith γ-synuclein might play an important though not completely understood rolein the pathogenesis of glaucoma. It is a matter of further investigation to findthe normal function of synucleins in HTM and RGC, and to establish the role ofγ-synuclein–myocilin interaction in glaucoma development.

ACKNOWLEDGMENTS

The authors thank Neeraj Agarwal (University of Oklahoma Health SciencesCenter) for RGC-5 cells, Dan Stammer (University of Arizona, Tucson) for myocilin


antibodies and primary culture of HTM cells, Mike Fautsch (Mayo Clinic Collegeof Medicine, Rochester, MN) for myocilin antibodies #651 and #653, and DenisSelkoe (Harvard Medical School) for R1735 sAPP antirabbit serum. The culture ofHT22 cells was provided by Dr. B. W. Festoff. Supported by National Institute ofHealth research grant EY 13784-03, grants from Glaucoma Foundation, Alzheimer’sDisease Research Center (ADRC), and Reeves Foundation.

REFERENCES

Adam, M. F., Belmouden, A., Binisti, P., Brezin, A. P., Valtot, F., Bechetoille, A., Dascotte, J. C., Copin,B., Gomez, L., Chaventre, A., Bach, J.-F., and Garchon, H.-J. (1997). Recurrent mutations in a singleexon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familialopen-angle glaucoma. Hum. Mol. Gen. 6:2091–2097.

Allingham, R. R., Wiggs, J. L., De La Paz, M. A., Vollrath, D., Tallett, D. A., Broomer, B., Jones, K. H.,Del Bono, E. A., Kern, J., Patterson, K., Haines, J. L., and Pericak-Vance, M. A. (1998). Gln368STOPmyocilin mutation in families with late-onset primary open angle glaucoma. Invest. Ophthalmol. Vis.Sci. 39:2288–2295.

Alward, W. L. M., Fingert, J. H., Coote, M. A., Johnson, A. T., Lerner, S. F., Junqua, D., Durcan, F. J.,McCartney, P. J., Mackey, D. A., Sheffield, V. C., and Stone, E. M. (1998). Clinical features associatedwith mutations in the chromosome 1 open-angle glaucoma gene (GLC1A). N. Engl. J. Med. 338:1022–1027.

Alward, W. L., Kwon, Y. H., Khanna, C. L., Johnson, A. T., Hayreh, S. S., Zimmerman, M. B., Narkiewicz,J., Andorf, J. L., Moore, P. A., Fingert, J. H., Sheffield, V. C., and Stone, E. M. (2002). Variations inthe myocilin gene in patients with open-angle glaucoma. Arch. Ophthalmol. 120:1189–1197.

Brenz Verca, M. S., Bahi, A., Boyer, F., Wagner, G. C., and Dreyer, J. L. (2003). Distribution of alpha-and gamma-synucleins in the adult rat brain and their modification by high-dose cocaine treatment.Eur. J. Neurosci. 18:1923–1938.

Bross, P., Corydon, T. J., Andresen, B. S., Jorgensen, M. M., Bolund, L., and Gregersen, N. (1999). Proteinmisfolding and degradation in genetic diseases. Hum. Mutat. 14:186–198.

Buchman, V. L., Hunter, H. J., Pinon, L. G., Thompson, J., Privalova, E. M., Ninkina, N. N., and Davies,A. M. (1998a). Persyn, a member of the synuclein family, has a distinct pattern of expression in thedeveloping nervous system. J. Neurosci. 18:9335–9341.

Buchman, V. L., Adu, J., Pinon, L. G., Ninkina, N. N., and Davies, A. M. (1998b). Persyn, a member ofthe synuclein family. Nat. Neurosci. 1:101–103.

Caballero, M., Rowlette, L. L., and Borras, T. (2000). Altered secretion of a TIGR/MYOC mutant lackingthe olfactomedin domain. Biochim. Biophys. Acta 1502:447–460.

Caballero, M., and Borras, T. (2001). Inefficient processing of an olfactomedin-deficient myocilin mu-tant: Potential physiological relevance to glaucoma. Biochem. Biophys. Res. Commun. 282:662–670.

Clark, A. F., Steely, H. T., Dickerson, J. E., Jr., English-Wright, S., Stropki, K., McCartney, M. D., Jacobson,N., Shepard, A. R., Clark, J. I., Matsushima, H., Peskind, E. R., Leverenz, J. B., Wilkinson, C. W.,Swiderski, R. E., Fingert, J. H., Sheffield, V. C., and Stone, E. M. (2001). Glucocorticoid inductionof the glaucoma gene MYOC in human and monkey trabecular meshwork cells and tissues. Invest.Ophthalmol. Vis. Sci. 42:1769–1780.

Clayton, D. F., and George, J. M. (1999). Synucleins in synaptic plasticity and neurodegenerative disorders.J. Neurosci. Res. 58(1):120–129.

Duda, J. E., Shah, U., Arnold, S. E., Lee, V. M., and Trojanowski, J. Q. (1999). The expression of alpha-,beta-, and gamma-synucleins in olfactory mucosa from patients with and without neurodegenerativediseases. Exp. Neurol. 160:515–522.

Farkas, R. H., Qian, J., Goldberg, J. L., Quigley, H. A., and Zack, D. J. (2004). Gene expression profilingof purified rat retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 45:2503–2513.

Fautsch, M. P., and Johnson, D. H. (2001). Characterization of myocilin–myocilin interactions. Invest.Ophthalmol. Vis. Sci. 42:2324–2331.

Fautsch, M. P., Vrabel, A. M., Peterson, S. L., and Johnson, D. H. (2004). In vitro and in vivo characteri-zation of disulfide bond use in myocilin complex formation. Mol. Vis. 10:417–425.

Fingert, J. H., Heon, E., Liebmann, J. M., Yamamoto, T., Craig, J. E., Rait, J., Kawase, K., Hoh, S. T., Buys,Y. M., Dickinson, J., Hockey, R. R., Williams-Lyn, D., Trope, G., Kitazawa, Y., Ritch, R., Mackey,


D. A., Alward, W. L., Sheffield, V. C., and Stone, E. M. (1999). Analysis of myocilin mutations in1703 glaucoma patients from five different populations. Hum. Mol. Genet. 6:2091–2097.

Friedman, J. S., and Walter, M. A. (1999). Glaucoma genetics, present and future. Clin. Genet. 55:71–79.Fung, K. M., Rorke, L. B., Giasson, B., Lee, V. M., and Trojanowski, J. Q. (2003). Expression of alpha-,

beta-, and gamma-synuclein in glial tumors and medulloblastomas. Acta Neuropathol. 106:167–175.Giasson, B. I., Forman, M. S., Higuchi, M., Golbe, L. I., Graves, C. L., Kotzbauer, P. T., Trojanowski, J.

Q., and Lee, V. M. (2003). Initiation and synergistic fibrillization of tau- and alpha-synuclein. Science300:636–640.

Gobeil, S., Rodrigue, M. A., Moisan, S., Nguyen, T. D., Polansky, J. R., Morissette, J., and Raymond, V.(2004). Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causingmyocilin mutants. Invest. Ophthalmol. Vis. Sci. 45:3560–3567.

Iwai, A., Masliah, E., Yoshimoto, M., Ge, N., Flanagan, L., de Silva, H. A., Kittel, A., and Saitoh, T. (1995).The precursor protein of non-A beta component of Alzheimer’s disease amyloid is a presynapticprotein of the central nervous system. Neuron 14:467–475.

Jacobson, N., Andrews, M., Shepard, A. R., Nishimura, D., Searby, C., Fingert, J. H., Hageman, G., Mullins,R., Davidson, B. L., Kwon, Y. H., Alward, W. L., Stone, E. M., Clark, A. F., and Sheffield, V. C. (2001).Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshworkcells and in aqueous humor. Hum. Mol. Gen. 10:117–125.

Ji, H., Liu, Y. E., Jia, T., Wang, M., Liu, J., Xiao, G., Joseph, B. K., Rosen, C., and Shi, Y. E. (1997).Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing.Cancer Res. 57:759–764.

Jiang, Y., Liu, Y. E., Goldberg, I. D., and Shi, Y. E. (2004). Gamma synuclein, a novel heat-shock protein-associated chaperone, stimulates ligand-dependent estrogen receptor alpha signaling and mammarytumorigenesis. Cancer Res. 64:4539–4546.

Johnson, A. T., Alward, W. L. M., Sheffield, V. C., and Stone, E. M. (1996). Genetics and glaucoma. InRitch, R., Shields, M. B., and Krupin, T. (eds.), The Glaucomas, CV Mosby, St. Louis, pp. 39–54.

Jurynec, M. J., Riley, C. P., Gupta, D. K., Nguyen, T. D., McKeon, R. J., and Buck, C. R. (2003). TIGR isupregulated in the chronic glial scar in response to central nervous system injury and inhibits neuriteoutgrowth. Mol. Cell. Neurosci. 23:69–80.

Kirstein, L., Cvekl, A., Chauhan, B. K., and Tamm, E. R. (2000). Regulation of human myocilin/TIGRgene transcription in trabecular meshwork cells and astrocytes: Role of upstream stimulatory factor.Genes Cells 5:661–676.

Krishnamoorthy, R. R., Agarwal, P., Prasanna, G., Vopat, K., Lambert, W., Sheedlo, H. J., Pang, I. H.,Shade, D., Wordinger, R. J., Yorio, T., Clark, A. F., and Agarwal, N. (2001). Characterization of atransformed rat retinal ganglion cell line. Mol. Brain Res. 86:1–12.

Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J. T., Schols,L., and Riess, O. (1998). Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’sdisease. Nat. Genet. 18:106–108.

Kulkarni, N. H., Karavanich, C. A., Atchley, W. R., and Anholt, R. R. (2000). Characterization anddifferential expression of a human gene family of olfactomedin-related proteins. Genet. Res. 76:41–50.

Lavedan, C., Leroy, E., Dehejia, A., Buchholtz, S., Dutra, A., Nussbaum, R. L., and Polymeropoulos,M. H. (1998). Identification, localization and characterization of the human gamma-synuclein gene.Hum. Genet. 103:106–112.

Lindersson, E., Beedholm, R., Hojrup, P., Moos, T., Gai, W., Hendil, K. B., and Jensen, P. H. (2004).Proteasomal inhibition by alpha-synuclein filaments and oligomers. J. Biol. Chem. 279:12924–12934.

Ling, L., Cao, Z., and Goeddel, D. V. (1998). NF-kappaB-inducing kinase activates IKK-alpha by phos-phorylation of Ser-176. Proc. Natl. Acad. Sci. U.S.A. 95:3792–3797.

Liu, Y., and Vollrath, D. (2004). Reversal of mutant myocilin non-secretion and cell killing: Implicationsfor glaucoma. Hum. Mol. Genet. 13:1193–1204.

Mackey, D. A., Alward, W. L., Sheffield, V. C., and Stone, E. M. (1999). Analysis of myocilin mutationsin 1703 glaucoma patients from five different populations. Hum. Mol. Genet. 6:2091–2097.

Malinin, N. L., Boldinm, M. P., Kovalenko, A. V., and Wallach, D. (1997). MAP3K-related kinase involvedin NF-kappaB induction by TNF, CD95 and IL-1. Nature 385:540–544.

Mertts, M., Garfield, S., Tanemoto, K., and Tomarev, S. I. (1999). Identification of the region in the N-terminal domain responsible for the cytoplasmic localization of Myoc/Tigr and its association withmicrotubules. Lab. Invest. 79:1237–1245.

Michels-Rautenstrauss, K. G., Mardin, C. Y., Budde, W. M., Liehr, T., Polansky, J., Nguyen, T., Timmerman,V., Van Broeckhoven, C., Naumann, G. O., Pfeiffer, R. A., and Rautenstrauss, B. W. (1998). Juvenileopen-angle glaucoma: Fine mapping of the myocilin gene to 1q243–q252 and mutation analysis. Hum.Genet. 102:103–106.


Michels-Rautenstrauss, K., Mardin, C., Wakili, N., Junemann, A. M., Villalobos, L., Mejia, C., Soley,G. C., Azofeifa, J., Ozbey, S., Naumann, G. O., Reis, A., and Rautenstrauss, B. (2002). Novel mu-tations in the MYOC/GLC1A gene in a large group of glaucoma patients. Hum. Mutat. 20:479–480.

Nakaya, K. (1994). Localization of phosphoneuroprotein 14 (PNP 14) and its mRNA expression in ratbrain determined by immunocytochemistry and in situ hybridization. Brain. Res. Mol. Brain. Res.27:81–86.

Nguyen, T. D., Chen, P., Huang, W. D., Chen, H., Johnson, D., and Polansky, J. R. (1998). Gene structureand properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-inducedtrabecular meshwork cells. J. Biol. Chem. 273:6341–6350.

Noda, S., Mashima, Y., Obazawa, M., Kubota, R., Oguchi, Y., Kudoh, J., Minoshima, S., and Shimizu,N. (2000). Myocilin expression in the astrocytes of the optic nerve head. Biochem. Biophys. Res.Commun. 276:1129–1135.

O’Brien, E. T., Ren, X., and Wang, Y. (2000). Localization of myocilin to the Golgi apparatus in Schlemm’scanal cells. Invest. Ophthalmol. Vis. Sci. 41:3842–3849.

Ohlmann, A., Goldwich, A., Flugel-Koch, C., Fuchs, A. V., Schwager, K., and Tamm, E. R. (2003).Secreted glycoprotein myocilin is a component of the myelin sheath in peripheral nerves. Glia 43:128–140.

Pang, C. P., Leung, Y. F., Fan, B., Baum, L., Tong, W. C., Lee, W. S., Chua, J. K., Fan, D. S., Liu, Y., andLam, D. S. (2002). TIGR/MYOC gene sequence alterations in individuals with and without primaryopen-angle glaucoma. Invest. Ophthalmol. Vis. Sci. 43:3231–3235.

Park, S. M., Jung, H. Y., Kim, H. O., Rhim, H., Paik, S. R., Chung, K. C., Park, J. H., and Kim, J. (2002).Evidence that alpha-synuclein functions as a negative regulator of Ca(++)-dependent alpha-granulerelease from human platelets. Blood 100:2506–2514.

Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike, B.,Root, H., Rubenstein, J., Boyer, R., Stenroos, E. S., Chandrasekharappa, S., Athanassiadou, A.,Papapetropoulos, T., Johnson, W. G., Lazzarini, A. M., Duvoisin, R. C., Di Iorio, G., Golbe, L. I., andNussbaum, R. L. (1997). Mutation in the alpha-synuclein gene identified in families with Parkinson’sdisease. Science 276:2045–2047.

Quigley, H. A. (1996). Number of people with glaucoma worldwide. Br. J. Ophthalmol. 80:389–393.Royds, J. A., Parsons, M. A., Taylor, C. B., and Timperley, W. R. (1982). Enolase isoenzyme distribution

in the human brain and its tumors. J. Pathol. 137:37–49.Shepard, A. R., Jacobson, N., Fingert, J. H., Stone, E. M., Sheffield, V. C., and Clark, A. F. (2001). Delayed

secondary glucocorticoid rewponsiveness of MYOC in human trabecular meshwork cells. Invest.Ophthalmol. Vis. Sci. 42:3173–3181.

Shepard, A. R., Jacobson, N., Sui, R., Steely, T., Lotery, A. J., Stone, E. M., and Clark, A. F. (2003).Characterization of rabbit myocilin: Implications for human myocilin glycosylation and signal peptideusage. BMC Genet. 4:1–10.

Singleton, A. B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M., Peuralinna, T.,Dutra, A., Nussbaum, R., Lincoln, S., Crawley, A., Hanson, M., Maraganore, D., Adler, C., Cookson,M. R., Muenter, M., Baptista, M., Miller, D., Blancato, J., Hardy, J., and Gwinn-Hardy, K. (2003).alpha-Synuclein locus triplication causes Parkinson’s disease. Science 302:841.

Snyder, H., Mensah, K., Theisler, C., Lee, J., Matouschek, A., and Wolozin, B. (2003). Aggregated andmonomeric alpha-synuclein bind to the S6′ proteasomal protein and inhibit proteasomal function.J. Biol. Chem. 278:11753–11759.

Snyder, H. M., Mensah, K., Surgucheva, I., Festoff, B., Surguchov, A., and Wolozin, B. (2005).β-Synuclein prevents proteasomal inhibition by α-synuclein but not γ-synuclein. J. Biol. Chem.280:7562–7569.

Souza, J. M., Giasson, B. I., Lee, V. M., and Ischiropoulos, H. (2000). Chaperone-like activity of synucleins.FEBS Lett. 474:116–119.

Stamer, W. D., Roberts, B. C., Howell, D. N., and Epstein, D. L. (1998). Isolation, culture, and characteri-zation of endothelial cells from Schlemm’s canal. Invest. Ophthalmol. Vis. Sci. 39:1804–1812.

Stone, E. M., Fingert, J. H., Alward, W. L., Nguyen, T. D., Polansky, J. R., Sunden, S. L., Nishimura, D.,Clark, A. F., Nystuen, A., Nichols, B. E., Mackey, D. A., Ritch, R., Kalenak, J. W., Craven, E. R.,and Sheffield, V. C. (1997). The identification of a gene that causes primary open-angle glaucoma.Science 5300:668–670.

Surguchov, A., McMahon, B., Masliah, E., and Surgucheva, I. (2001a). Synucleins in ocular tissues.J. Neurosci. Res. 65:68–77.

Surguchov, A., Palazzo, R. E., and Surgucheva, I. (2001b). Gamma synucleins: Subcellular localization inneuronal and non-neuronal cells and effect on signal transduction. Cell Motil. Cytoskeleton 49:218–228.


Surgucheva, I., McMahon, B., Ahmed, F., Tomarev, S., Wax, M. B., and Surguchov, A. (2002). Synucleinsin glaucoma: Implication of γ-synuclein in glaucomatous alterations in the optic nerve. J. Nerosci.Res. 68:97–106.

Swiderski, R. E., Ying, L., Cassell, M. D., Alward, W. L., Stone, E. M., and Sheffield, V. C. (1999).Expression pattern and in situ localization of the mouse homologue of the human MYOC (GLC1A)gene in adult brain. Brain Res. Mol. Brain Res. 68:64–72.

Tamm, E. R. (2002). Myocilin and glaucoma: Facts and ideas. Prog. Retin. Eye Res. 21:395–428.Tanji, K., Mori, F., Imaizumi, T., Yoshida, H., Matsumiya, T., Tamo, W., Yoshimoto, M., Odagiri, H.,

Sasaki, M., Takahashi, H., Satoh, K., and Wakabayashi, K. (2002). Upregulation of alpha-synucleinby lipopolysaccharide and interleukin-1 in human macrophages. Pathol. Int. 52:572–577.

Tompkins, M. M., Gai, W. P., Douglas, S., and Bunn, S. J. (2003). Alpha-synuclein expression localizes tothe Golgi apparatus in bovine adrenal medullary chromaffin cells. Brain Res. 984:233–236.

Torrado, M., Trivedi, R., Zinovieva, R., Karavanova, I., and Tomarev, S. I. (2002). Optimedin: A novelolfactomedin-related protein that interacts with myocilin. Hum. Mol. Genet. 11:1291–1301.

Tripathi, B. J., and Tripathi, R. C. (1989). Neural crest origin of human trabecular meshwork and itsimplications for the pathogenesis of glaucoma. Am. J. Ophthalmol. 107:583–590.

Ueda, J., Wentz-Hunter, K. K., Cheng, E. L., Fukuchi, T., Abe, H., and Yue, B. Y. (2000). Ultrastructurallocalization of myocilin in human trabecular meshwork cells and tissues. J. Histochem. Cytochem.48:1321–1330.

Wentz-Hunter, K., Ueda, J., and Yue, B. Y. (2002). Protein interactions with myocilin. Invest. Ophthalmol.Vis. Sci. 43:176–182.

Wentz-Hunter, K., Shen, X., and Yue, B. Y. (2003). Distribution of myocilin, a glaucoma gene product, inhuman corneal fibroblasts. Mol. Vis. 9:308–314.

Wentz-Hunter, K., Shen, X., Okazaki, K., Tanihara, H., and Yue, B. Y. (2004). Overexpression of myocilinin cultured human trabecular meshwork cells. Exp. Cell. Res. 297:39–48.

Wordinger, R. J., Lambert, W., Agarwal, R., Talati, M., and Clark, A. F. (2000). Human trabecularmeshwork cells secrete neurotrophins and express neurotrophin receptors (Trk). Invest. Ophthal.Vis. Sci. 41:3833–3841.

WuDunn, D. (2002). Genetic basis of glaucoma. Curr. Opin. Ophthalmol. 13:55–60.Zhou, Z., and Vollrath, D. (1999). A cellular assay distinguishes normal and mutant TIGR/myocilin

protein. Hum. Mol. Genet. 8:2221–2228.

Interaction of Myocilin with γ-Synuclein Affects Its Secretion and Aggregation

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