Synucleins in Ocular Tissues

Andrei Surguchov,1* Belinda McMahan,1 Eleizer Masliah,2 and Irina Surgucheva1

1Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri2Department of Pathology and Neuroscience, the University of California at San Diego, La Jolla,California

Synucleins are small proteins associated with neurode-generative diseases and some forms of cancer. Moststudies of this group of proteins have been directed tothe elucidation of their role in the brain and their connec-tion to the formation of depositions in brain tissues. Herewe describe the localization of different types of synucle-ins in ocular tissues. By Western blot analysis, all mem-bers of the synuclein family are found in the retina andoptic nerve, where their relative ratio varies. The data onimmunohistochemical staining show that different mem-bers of the synuclein family have different localizations inocular tissues. a-Synucleins and b-synucleins arepresent predominantly in the inner plexiform layer,whereas g-synuclein is in the nerve fiber layer. In trans-genic mice overexpressing a-synuclein, a different pat-tern of localization depending on the promoter used forthe expression was observed. In Alzheimer’s diseasepatients, immunohistochemical staining for g-synucleinrevealed the loss of immunoreactivity in the nerve fiberlayer and the nerve fiber layer and the appearance ofimmunopositive cells in or near the outer nuclear layer.We conclude that, in mature eyes, synucleins are presentpredominantly in the retina and optic nerve, and theimmunoreactivity of g-synuclein changes specifically inthe retina of Alzheimer’s disease patients. In transgenicmice overexpressing a-synuclein, immunopositive de-posits in the optic nerve and accumulation of immuno-reactivity in specific retinal cells were found. J. Neurosci.Res. 65:68–77, 2001. © 2001 Wiley-Liss, Inc.

Key words: synucleins; retina; optic nerve; developmen-tal regulation; transgenic mice

INTRODUCTIONSynucleins are small proteins (14.5–20 kDa) that have

been implicated specifically in several diseases, e.g., Alzhei-mer’s disease (AD; Ueda et al., 1993; Spillantini et al., 1997;Mezey et al., 1998; Lippa et al., 1999), Parkinson’s disease(PD; Polymeropoulos et al., 1997; Krueger et al., 1998),Lewy body dementia (Trojanowski et al., 1998), and breastcancer (Ji et al., 1997; Jia et al., 1998). In the first studies, theywere found to be abundant in the neuronal cytosol andpresynaptic terminals (Maroteaux, 1988; Iwai et al., 1995).However, later it was shown that their expression is notlimited to neural tissues. For example, high levels ofg-synuclein were found in advanced breast carcinoma, ovar-ian tumor, and skin (Ji et al., 1997; Jia et al., 1998; Lavedanet al., 1998; Ninkina et al., 1998b).

a-Synuclein is a presynaptic protein found abun-dantly in the human brain and, at lower levels, in othertissues (Ueda et al., 1993; Jakes et al., 1994; Duda et al.,1999). It contains 140 amino acids and has five imperfectrepeats, with a consensus sequence KTKEGV in theN-terminal half. a-Synuclein binds to small unilamellarphospholipid vesicles containing acidic phospholipids butnot to vesicles with a net neutral charge (Davidson et al.,1998). Lipid binding is accompanied by an increase inalpha-helicity from 3% to approximately 80%. These ob-servations are consistent with a role in vesicle function atthe presynaptic terminal. a-Synuclein, or its fragment,may be responsible for the conversion of soluble Ab to theAb amyloid characteristic of AD (Jensen et al., 1997).Two separate missense mutations (A30P and A53T) havebeen identified in very rare cases of PD with autosomaldominant inheritance (Polymeropoulos et al., 1997;Kruger et al., 1998). However, in many other cases offamilial PD the association with these mutations was notfound (Chan et al., 1998; Farrer et al., 1998; Vaughan etal., 1998a,b). No mutation was found in a group ofpatients with AD (Campion et al., 1995). Knockout micethat lack the gene for a-synuclein exhibit a subtle pheno-type that implies that the protein can act as a negativeregulator of dopamine release (Abeliovich et al., 2000).Overexpression of human a-synuclein causes dopamineneuron death in rat primary culture and immortalizedmesencephalon-derived cells (Zhou et al., 2000). Recentevidence shows that a-synuclein can aggregate and formamyloid-like fibrils when present at high concentration(El-Agnaf et al., 1998a,b; Hashimoto et al., 1998), insolution with heavy metals such as copper (Paik et al.,1999) or iron (Ostrerova et al., 2000), or during oxidativestress (Hashimoto et al., 1999).

Contract grant sponsor: NEI; Contract grant number: EY 02687; Contractgrant sponsor: NIH; Contract grant number: AG 5131; Contract grantnumber: AG 10869; Contract grant sponsor: The Glaucoma Foundation;Contract grant number: QB42308; Contract grant sponsor: Carl MarshallReeves and Mildred Almen Reeves Foundation; Contract grant sponsor:ADRC; Contract grant number: 99-6403.

*Correspondence to: Andrei Surguchov, Department of Ophthalmologyand Visual Sciences, Washington University, Campus Box 8096, 660 S.Euclid Ave., St. Louis, MO 63110. E-mail: surguchov@vision.wustl.edu

Received 12 January 2001; Revised 27 February 2001; Accepted 28 Feb-ruary 2001

Journal of Neuroscience Research 65:68–77 (2001)

© 2001 Wiley-Liss, Inc.

According to recent observations, a-synuclein nitra-tion plays an important role in its synuclein aggregation,oxidative damage, and formation of a-synuclein filaments(Giasson et al., 2000; Duda et al., 2000). Several observa-tions demonstrate that a-synuclein inhibits phospholipaseD2 (PLD2), which produces phosphatidic acid by hydro-lysis of phosphatidylcholin (Exton, 2000; Oh et al., 2000)and protein kinase C (Ostrerova et al., 1999).

b-Synuclein (PNP14) is abundant in the brain, butsmall amounts of the protein have been detected in Sertolicells of neuroendocrine tissues (Shibayama-Imazu et al.,1998) and other tissues (Shibayama-Imazu et al., 1993). Incontrast to the case for a-synuclein, there are no data onthe role of b-synuclein in any diseases, except for onerecent publication demonstrating a possible role ofb-synuclein in PD and dementia with Lewy body (Galvinet al., 1999).

g-Synuclein (BCSG1, persyn) has a distinct patternof expression in the peripheral and central nervous sys-tems. In addition, it is present in the skin, i.e., stratumgranulosum of the epidermis, and is overexpressed inbreast and ovarian cancer (Ji et al., 1997; Ninkina et al.,1998a,b; Lavedan et al., 1998). g-Synuclein influencesneurofilament network integrity in sensory neurons andmay be involved in the modulation of the keratin network(Buchman et al., 1998; Ninkina et al., 1998b). In a recentpaper, the antibody to g-synuclein was shown to detectpreviously unrecognized axonal spheroid-like lesions inthe hippocampal dentate molecular layer of PD and Lewybody dementia (Galvin et al., 1999).

Synoretin (GenBank accession No. bankit31202) is asecond member of the g-synuclein subfamily initiallycloned from a bovine retina library. A similar cDNAsequence was isolated by amplification of a human cDNAlibrary (Surguchov et al., 1999). However, our attempts toclone the human gene by hybridization failed, so in furtherexperiments we used only bovine tissues to characterizesynoretin localization. It is expressed extensively in theretina and optic nerve, can be phosphorylated by Gprotein-coupled receptor kinases, and may affect somesignal transduction pathways (Surguchov et al., 1999; Pro-nin et al., 2000).

In spite of considerable information pointing to therole of synucleins in different human diseases, their normalfunctions remain elusive. The data on the effect of over-expression of synucleins in different cell types are contro-versial. Overexpression of wild-type and mutant forms ofa-synuclein, but not g-synuclein, in cultured neurons,causes apoptosis (Saha et al., 2000). Although in severalpublications toxicity of overexpressed a-synuclein wasobserved, others have not found any significant effect ofsynuclein overexpression on cell viability (O’Mally andJensen, 2000; Stefanova et al., 2000).

Here we demonstrate that synucleins are expressed inocular tissues, predominantly in the retina and the opticnerve. We also describe changes in g-synuclein localiza-tion in the retina of AD patients and the staining pattern of

a-synuclein in transgenic mice overexpressing this proteinunder different promoters.

MATERIALS AND METHODS

Antibodies

Rabbit polyclonal antisera were raised against keyholelimpet hemocyanin conjugates of synthetic peptides derivedfrom the predicted structure of human g-synuclein (Lavedan etal., 1998; Ninkina et al., 1998a) or synoretin (Surguchov et al.,1999). The following peptides were used: Ac-C-DLRPS-APQQEGEASKEK-amid (g-synuclein; amino acids 99–115)and Ac-C-ALKQPVPPQEDEAAKAE-amid (synoretin; aminoacids 99–115). The immunogenic peptide for synoretin was twoamino acids closer to the C-terminus of the protein compared tothe one used previously. Antisera were screened by ELISA andWestern blots using the immunizing peptides and retina extract,respectively. Antibodies were then affinity purified by reactingwith immunizing peptide immobilized on cyanogen bromide-activated Sepharose 4B. Antibodies for g-synuclein were diluted1:4,000 for Western blotting and 1:2,500 for immunohisto-chemical (IHC) staining; the dilution of antibodies for synoretinwas 1:3,500 for Western blotting and 1:500 for IHC staining. Incontrol experiments with antibodies to synoretin andg-synuclein preadsorbed with corresponding antigen, no stain-ing was observed. We used as antibodies for a-synuclein rabbitpolyclonal antiserum produced by Chemicon International Inc.(Temecula, CA) diluted 1:2,000 for Western blotting and1:1,500 for IHC staining. For the staining of transgenic micesamples, monoclonal antibody LB509 for a-synuclein (Zymed,San Francisco, CA) was used. Affinity-purified polyclonal rabbitanti-b-synuclein antibody was purchased from Chemicon In-ternational Inc. and diluted 1:2,000 for both Western blottingand IHC staining. For IHC staining, biotinylated secondaryantibody was identified by reacting with the streptavidin-peroxidase conjugate (Vector Laboratories, Burlingame, CA).For Western blotting, secondary antibodies were conjugatedwith peroxidase, dilution 1:50,000 (Pierce, Rockford, IL).

Recombinant Synucleins

a-, b-, and g-synucleins were gift from Dr. J. Tro-janowski. Recombinant synoretin was obtained as describedpreviously (Surguchov et al., 1999).

Western Blots

Human retinas, optic nerves, and other ocular tissues weredissected from eyes obtained from Mid-America TransplantServices (St. Louis, MO). Retinas were disintegrated by soni-cation, and other tissues were disrupted in a Turrax tissuehomogenizer as described earlier (Surguchov et al., 1999). Totalprotein was measured by the Bradford method (Pierce). Humanbrain tissue homogenate was from Clontech Laboratories, Inc.(Palo Alto, CA). Bovine eyes were received from a local slaugh-terhouse; extracts from bovine ocular tissues were preparedsimilarly to those from humans. Fifteen to twenty micrograms oftotal protein extract was loaded on a 12% polyacrylamide gel.After electrophoresis, proteins were transferred onto NC0.45 mM (BioRad, Hercules, CA) and blots were treated asdescribed earlier (Surguchov et al., 1999). To demonstrate the

Synucleins in Ocular Tissues 69

efficiency of antibody for each of the synucleins, the experi-ments were carried out as follows. Each purified recombinantsynuclein was run on polyacrylamide gel and blotted on nitro-cellulose 0.45 mM, and then pieces of nitrocellulose were cut offand incubated with each of the corresponding antibodies sepa-rately. The results are shown in Figure 1C.

IHC Staining

Human eyes (donor ages 53–71 years) with no history ofdisease were obtained from eye banks throughout the UnitedStates. The number of normal human eyes that we used forstaining was 12. From 8 to 12 sections were prepared andanalyzed from the central part of the retina and from the opticnerve. Representative images of the retina and optic nerve areshown in Figure 1. Eyes from AD patients were from donorsaged 71 or 72 years who died of respiratory failure withouthistory of eye diseases. The interval between death and fixationfor all donors was less than 4 hr. After enucleation, eyes were

washed in phosphate-buffered saline (PBS) containing 0.1%glycine and processed for paraffin embedding. Slices of 5 mmwere cut and placed on silane-coated slides. Before immuno-staining, slides were deparaffinized and incubated for 1 hr in PBSglycine at room temperature to reduce nonspecific binding.Slides were preincubated with 5% milk for 30 min, rinsed, andincubated with primary antibodies for 30 min. Biotinylatedsecondary antibody was placed on the sections and incubated for30 min, washed with PBS, and reacted with the streptavidin-peroxidase conjugate (Vector Laboratories) for 30 min. Thebound antibody-peroxidase complexes on the sections werevisualized using a 3,3-diaminobenzidine tetrahydrochloride(DAB) substrate solution consisting of 1.5 mg DAB and 50 ml of30% hydrogen peroxide in 10 ml of 0.1 M Tris, pH 7.6. Thesections were incubated in the dark until brown staining ap-peared, washed in PBS, counterstained with hematoxylin, de-hydrated, and coverslipped with Permount. Control sectionswere run in parallel, omitting only the primary antibody.

Fig. 1. Electrophoresis in 12% polyacrylamide gel. A: Coomassie stain-ing: lane 1, ladder; lanes 2–5: a-, b-, and g-synuclein, and synoretin,respectively. B: Immunoblot stained with synoretin antibody: lanes1–4, recombinant a-, b-, g-synuclein, and synoretin, respectively.C: Immunoblot with (from left to right) a-, b-, g-synuclein, andsynoretin stained with corresponding antibodies. D: Immunoblot of

extracts from different human eye (top three sections) tissues stainedwith a-, b-, g-synuclein, and synoretin antibodies (from top to bot-tom) and bovine eye tissues (bottom section). R, retina; ON, opticnerve; L, lens; Co, cornea; Ir, iris; RPE, retinal pigment epithelium;Rec, recombinant synoretin; Br, brain.

70 Surguchov et al.

We also analyzed 10 bovine eyes, which were treatedsimilarly to human eyes. From 8 to 12 sections were preparedand analyzed from the central part of the retina and from theoptic nerve. The interval between death and fixation for bovinesamples was less than 1 hour.

Transgenic Mice

All procedures with mice were in accordance with theguidelines published in the NIH Guide for the Care and Use ofLaboratory Animals (National Institutes of Health PublicationNo. 85-23) and the principles presented in the “Guidelines forthe Use of Animals in Neuroscience Research” by the Societyfor Neuroscience.

a-Synuclein transgenic mice were generated as previouslydescribed (Masliah et al., 2000). The eyes of two groups oftransgenic mice were analyzed. In one, human wild-typea-synuclein was expressed under the regulatory control of theplatelet-derived growth factor (PDGF)-b promoter. These miceshow inclusion body formation in the brain and motor anddopaminergic deficits. Two lines of mice from this group wereinvestigated, the low expresser (line A) and the high expresser(line D). In addition, a-synuclein immunostaining was alsoinvestigated in the eyes of transgenic mice expressing wild-typea-synuclein under the control of the thy-1 promoter wereanalyzed. Briefly, the murine thy-1 expression cassette (providedby Dr. H. van der Putten, Ciba-Geigy, Basel, Switzerland) wasused to generate the transgenic mice. The wild-type humana-synuclein cDNA fragment was generated by RT-PCR from

human brain mRNA. The a-synuclein cDNA fragment wasinserted into the mthy-1 expression cassette between exons 2and 4, purified, and microinjected into one-cell embryos(C57BL/6 3 DBA/2 F1) according to standard procedures.Genomic DNA was extracted from tail biopsies and analyzedwith PCR amplification. Transgenic expresser lines were main-tained by crossing heterozygous transgenic mice with nontrans-genic C57BL/6 3 DBA/2 F1 breeders. All transgenic micewere heterozygous with respect to the transgene. Nontransgeniclittermates served as controls.

Mice were anesthetized with chloral hydrate and flush-perfused transcardially with 0.9% saline. Eyes and brains wereremoved and divided sagittally. One hemibrain was postfixed inphosphate-buffered 4% paraformaldehyde (pH 7.4) at 4°C for48 hr for vibratome sectioning; the other was snap frozen andstored at 270°C for RNA or protein analysis. Five transgenicmice with each of these two promoters and five aged-matchedcontrols were used for immunohistochemical staining. Repre-sentative images of the retina from wild-type and transgenicmice are shown in Figure 4.

RESULTSSpecificity of Antisynuclein Antibodies

Although different isoforms of synucleins have sim-ilar amino acid sequences at their N-termini, antibodiescan be raised that distinguish all members of the synucleinfamily. This is due to the presence of fragments in theiramino acid sequences located either in C-terminal parts or

Fig. 2. Immunohistochemical localization of a-, b-, and g-synucleinsin human retina (A–C) and optic nerve (D–F). Synucleins were de-tected using DAB reagent (peroxidase; brown); tissues were counter-stained with hematoxylin (blue). Sections were stained with antibody

that recognizes a-synuclein (A,D), b-synuclein (B,E), or g-synuclein(C,F). NFL, nerve fiber layer; IPL, inner plexiform layer; INL, innernuclear layer; ONL, outer nuclear layer; IS, inner segment of photo-receptor cells. Scale bars 5 50 mm.

Synucleins in Ocular Tissues 71

between C-termini and central parts of the molecule, e.g.,between amino acid residues 99 and 114, where a-, b-,and g-synucleins have variable sequence (Lavedan et al.,1998; Clayton and George, 1998). In these fragments, allmembers of the family have more than 50% difference inthe amino acid sequence of a 15-amino-acid fragment thatcan be selected for peptide antibody production (Surgu-chov et al., 1999). The specificity of antibodies to a-, b-,and g-synuclein and synoretin was demonstrated by West-ern blot analysis using recombinant proteins. Figure 1Ashows Coomassie staining of purified recombinant

synucleins used for antibody specificity characterization.From Figure 1B, one can see that antibody to synoretindoes not recognize a-, b-, and g-synucleins under con-ditions when these proteins are recognized by their cor-responding antibodies (Fig. 1C). In addition, antibodies toa-, b-, and g-synucleins do not recognize recombinantsynoretin (Fig. 1D, lane 7). High specificity of antibodiesto a-, and b-synucleins raised to a unique peptide regionat the C-terminus was shown earlier on immunoblotsusing native and recombinant a- and b-synuclein (Jakes etal., 1994).

Tissue Specificity of Synucleins in the EyeThe presence of synucleins in human ocular tissues

was studied by Western blot analysis (Fig. 1D).a-synuclein, b-synuclein, and g-synuclein were found inthe retina (Fig. 1D, R) and optic nerve (Fig. 1D, ON),where their relative ratios vary. In addition, two weakbands, one of which has the same size as a retinal protein,were found in the iris (Fig. 1D, Ir) when stained byb-synuclein antibodies. Antibodies to a- and b-synucleinstained a major band in the brain (Fig. 1C, Br) comparedto the retina (Fig. 1C, R). In bovine eye tissues, synoretinis more abundant in the optic nerve and retina (Fig. 1D, Rand ON) compared to the brain (Fig. 1D, Br) and is almostabsent in other tissues, except for small amounts in thecornea and iris. Recombinant synoretin shows the samegel mobility as its native counterpart [compare electro-phoretic mobility of recombinant synoretin (Fig. 1D, Rec)to that of the protein in the retina (Fig. 1D, R) and opticnerve (Fig. 1D, ON)].

Immunohistochemical Localization of Synucleinsin the Retina and Optic Nerve

Each of the members of synuclein family possesses adistinct pattern of localization in human retina and opticnerve. However, the pattern of a-synuclein immunore-activity is more similar to that of b-synuclein (Fig. 2A, B),whereas the staining pattern of g-synuclein in humanretina is different (Fig. 2C) and resembles the localizationof synoretin in bovine retina (Fig. 3A). The majority of a-and b-synuclein is in the inner plexiform layer (IPL;brown staining; Fig. 2A,B). In addition, a-synuclein im-munoreactivity is found in individual cells (presumably,amacrine cells), located on the inner margin of the innernuclear layer (INL; Fig. 2A, arrows). Cells positive forb-synuclein are seen throughout the INL consisting basi-cally of nuclei of interneurons (amacrine, bipolar, andhorizontal cells; Fig. 2B, arrows). g-Synuclein immuno-reactivity is concentrated in the nerve fiber layer (NFL)and individual cells in the ganglion cell layer (GCL; Fig.2C, arrows). In human optic nerve, a-, b-, andg-synuclein staining was found on the cell periphery (Fig.2D–F); in addition, intracellular staining of b-synucleinwas revealed in individual cells (Fig. 2E, arrows).

In bovine retina, synoretin is localized in the NFL(Fig. 3A), as does its human ortholog, g-synuclein inhuman retina (Fig. 2C). However, in contrast tog-synuclein, synoretin is also present in the inner segment

Fig. 3. Immunohistochemical localization of synoretin in bovine retina(A) and optic nerve (B). Synoretin immunoreactivity was detectedusing 3,39-diaminobenzidine reagent (peroxidase; brown); tissues werecounterstained with hematoxylin (blue). Arrows in A show a subset ofcells stained by synoretin antibody in the inner nuclear layer. Abbre-viations and staining methods as described in the legend to Figure 2.Scale bars 5 50 mm.

72 Surguchov et al.

of photoreceptor cells. A weak staining was also observedin the IPL and outer plexiform layer (OPL) and in indi-vidual cells of INL of bovine retina (Fig. 3A, arrows). Inbovine optic nerve, synoretin immunoreactivity is locatedin elongated cells, presumably axons of retinal ganglioncells (Fig. 3B).

a-Synuclein Transgenic MiceImmunohistochemical Staining of the Retina.

For the staining of the ocular samples from the transgenicmice, we used antibodies that recognize either human

(monoclonal Ab LB509 from Zymed) or mouse (Chemi-con) a-synuclein. The antibody specific to mouse but nothuman protein allowed us to identify those retinal cells inwhich endogenous a-synuclein is present (Fig. 4A,B). Asshown in Figure 4, immunopositive cells are located in ornear ganglion cell layer and in the inner segment (IS,arrowhead) of the photoreceptor cell layer. The localiza-tion of mouse endogenous a-synuclein in the retina issimilar but not identical to the localization of humanprotein (compare Fig. 2A and Fig. 4A,B). To study thelocalization of a-synuclein in the retina of transgenic mice

Fig. 4. Immunohistochemical staining of the retina from wild type(A,B) and a-synuclein transgenic mice (C,D). C: Thy promoter-driven a-synuclein expression. D: PDGF-b promoter-driven expres-sion. Both promoters are from human genes. A is age-matched non-transgenic control to C; B is age-matched nontransgenic control to D.

For other details concerning transgenic mice generation, see Masliah etal. [Science, 287, 1265–1269 2000]. A,B: Staining with antibodiesspecific for mouse a-synuclein (Chemicon). C,D: Staining with anti-bodies specific for human a-synuclein (monoclonal Ab LB509, fromZymed). Scale bars 5 50 mm.

Synucleins in Ocular Tissues 73

overexpressing this protein, we used two lines of mice inwhich a-synuclein was under different promoters: thy-1(Fig. 4C) and PDGF-b (Fig. 4D). We observed consid-erable differences in the staining patterns of wild-type (Fig.4A,B) and transgenic (Fig. 4C,D) mice: 1) In transgenicmice, a higher overall level of a-synuclein staining in thewhole retina was observed (more brown staining in Fig.4C and D compared to Fig. 4A and B). 2) In transgenicmice, the accumulation of a-synuclein immunoreactivitywas observed in a subset of cells located in the INL andGCL (Fig. 4C,D). A weaker staining was found in theinner segment of photoreceptor cells. The subset of im-munopositive cells in the INL of mice expressing trans-gene under the control of PDGF-b promoter looked

different (Fig. 4D, smaller, with processes) than in animalsexpressing transgene under the thy-1 promoter (staining oflarger cells, without processes).

Staining of the Optic Nerve. In contrast tonontransgenic controls (Fig. 5B), optic nerve of highexpresser humans, a-synuclein mice showed a-synuclein-immunopositive deposits (Fig. 5A). The accumulation ofsimilar deposits containing both wild-type and mutanta-synuclein in the brain tissues is considered to play acausal role in different neurodegenerative diseases. Furtherexperiments will be necessary to elucidate the role ofsynucleins in the optic nerve and their possible role in theneurodegenerative process and eye diseases.

Immunohistochemical Staining of Retina in ADPatients

In AD patients, IHC staining for g-synuclein re-vealed a partial loss of immunoreactivity in the NFL andthe appearance of immunopositive staining in a subset ofphotoreceptor cells and cells of OPL (Fig. 6A,B). At thesame time, we did not observe differences in the stainingpatterns for a- and b-synucleins.

DISCUSSIONOur results demonstrate the presence of all members

of the synuclein family in ocular tissues (Figs. 1–4). Theyare expressed predominantly in the retina and optic nerve,although smaller amounts are also present in the corneaand iris (Fig. 1D). In spite of the similarity in the aminoacid sequence of all four synucleins, their expression pat-tern is different and specific for each type of synuclein,suggesting differences in transcriptional regulation of theirgene expression. Western blot results showed that a- andb-synucleins are more abundant in the brain and arepresent in a lower amount in the retina (Fig. 1D).

Although the patterns of a- and b-synuclein immu-nostaining in the retina are similar to each other (Fig.2A,B), they are distinct from that of g-synuclein (Fig. 2C)and synoretin (Fig. 3A). a- And b-synucleins are locatedprimarily in the IPL, where synaptic contacts betweenganglion cells and interneurons take place. These findingsare in agreement with previous results on the location ofthese types of synucleins in synapses of neurons (Ueda etal., 1993; Jakes et al., 1994). It is curious that, among thetwo synaptic regions in the retina (IPL and OPL), the IPLcontains both a- and b-synuclein, whereas the OPL ap-pears to be devoid of synucleins. It is worth mentioningthat the IPL is largely composed of conventional synapses,whereas the OPL is primarily composed of ribbon synapses(Dowling, 1987).

Synaptic ribbons are specialized, electron-densestructures found in photoreceptors and other primary neu-rons that generate graded potentials, rather than actionpotentials. Other differences between these two types ofsynapses are also described, e.g., that both sequestering anddocking mechanisms of synaptic vesicles are different inribbon-containing neurons than in conventional, actionpotential-generating neurons (Balkema and Rizkalla,1996). In previous studies, a difference in protein compo-

Fig. 5. Immunohistochemical staining of optic nerve from transgenicmouse overexpressing a-synuclein (A) and wild-type mice (B).a-synuclein-immunopositive deposits were detected using DAB re-agent (peroxidase; brown); tissues were counterstained with hematox-ylin (blue). Scale bars 5 50 mm.

74 Surguchov et al.

sition of these two types of synapses has been reported;e.g., synapsin and rabphilin are present in conventional butabsent in ribbon synapses (Von Kriegstein et al., 1999).

An important difference between the localization ofa- and b-synucleins that may have functional implicationsis the presence of b-synuclein in the INL (Fig. 2A,B). Thislayer consists of nuclei of interneurons (amacrine, bipolar,and horizontal cells) and Muller cells. The presence ofanother member of the synuclein family, synoretin, in thenuclei of bipolar cells was shown previously (Surguchov etal., 1999). The localization of synuclein in the nuclei wasdemonstrated in the first paper describing this protein(Maroteaux et al., 1988), but since then it has not beenconfirmed.

According to IHC staining (Fig. 2C), the majority ofg-synuclein immunoreactivity occurs in the NFL. Thislayer is largely composed of ganglion cells whose collectiveaxons comprise the optic nerve, which conducts visualimpulses to the brain. The conclusion from the data onimmunolocalization of synucleins in the retina and opticnerve is that, in spite of their similarity in structure, thepattern of their localization in these types of tissues isdifferent, suggesting different mechanisms of gene expres-sion regulation and/or different pathways of protein tar-geting.

In transgenic mice overexpressing a-synuclein, ex-tensive accumulation of this protein in the retinal cells wasobserved. Because promoters of thy-1 and PDGF-b directthe expression predominantly in the GCL and NFL(Schmid et al., 1995; Dabin and Barnstable, 1995; Shep-pard et al., 1998; Mekada et al., 1998; Simpson et al.,1999), accumulation of a-synuclein in these retinal layers(Fig. 4C,D) was expected. Surprisingly, a very strong

expression of a-synuclein was also observed in the INL,presumably in glial cells. This may be explained by one oftwo mechanisms: 1) Expression of high amounts ofa-synuclein, possessing a toxic effect on cells (El-Agnaf etal., 1998a), affects glial cells and induces thy-1- andPDGF-b-driven transcription. Dabin and coworkers(1995) stated that ganglion cell death induces a glial re-sponse, including the expression of thy-1 in Muller cells,the main glial cell type in the retina. Their results suggestthat thy-1 is expressed by Muller cells following loss ofretinal neurons. According to these investigators, thy-1may have an important function during glial response toneuron death in retina. Although, in the transgenic micethat we used, no signs of retinal ganglion cell death werenoticed, overexpression of a toxic protein may induce thereaction described earlier (Dabin and Barnstable, 1995). 2)Another reason why we observe a pattern of thy-1- andPDGF-b-driven expression different from that describedfor these protein may be explained by the fact that someregulatory elements, e.g., silencers, in their genes may belocated a long distance from the coding region and, there-fore, are absent in the genetic constructs containing cor-responding promoters of their genes. Interestingly, thatoverexpression of a-synuclein does not affect the expres-sion of other members of the same family (results notshown).

In the retina of AD patients, IHC staining forg-synuclein revealed the reduction of immunoreactivity inthe NFL and the appearance of immunopositive cells in ornear the OPL (Fig. 6A–C). At the same time, no notice-able differences in the staining patterns for a- andb-synucleins were observed.

Fig. 6. Immunohistochemical staining of AD patient eyes with g-synuclein antibodies. A,B: Retinafrom two Alzheimer’s disease patients. C: Staining of age-matched control retina. a-Synucleinstaining was detected using DAB reagent (peroxidase; brown); tissues were counterstained withhematoxylin (blue). Scale bars 5 50 mm.

Synucleins in Ocular Tissues 75

This is the first report on the presence of synucleinsin ocular tissues. Further investigations will show whetherthey play an important role in neurodegeneration of theretina and optic nerve similar to that shown for the brainand in which eye diseases they may be involved.

ACKNOWLEDGMENTSWe thank Dr. John Trojanowski for recombinant

a-, b- and g-synucleins. We express our gratitude for Drs.Rosario Hernandez and Arthur Neufeld for many helpfulsuggestions concerning immunohistochemical staining.

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