Tauroursodeoxycholic Acid Prevents Retinal Degeneration in Transgenic P23H Rats

Retinal Cell Biology

Tauroursodeoxycholic Acid Prevents Retinal Degeneration in Transgenic P23H Rats Laura Ferna ´ ndez-Sa ´ nchez,1 Pedro Lax,1 Isabel Pinilla,2 Jose´ Martín-Nieto,1,3 and Nicola ´ s Cuenca1,3 PURPOSE. To evaluate the preventive effect of tauroursodeoxycholic acid (TUDCA) on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). METHODS. P23H line-3 rats were injected with TUDCA once a week from postnatal day (P)21 to P120, in parallel with vehicle-administered controls. At P120, functional activity of the retina was evaluated by electroretinographic (ERG) recording. The effects of TUDCA on the number, morphology, integrity, and synaptic connectivity of retinal cells were characterized by immunofluorescence confocal microscopy. RESULTS. The amplitude of ERG a- and b-waves was significantly higher in TUDCA-treated animals under both scotopic and photopic conditions than in control animals. In the central area of the retina, TUDCA-treated P23H rats showed threefold more photoreceptors than control animals. The number of TUNELpositive cells was significantly smaller in TUDCA-treated rats, in which photoreceptor morphology was preserved. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in TUDCA-treated P23H rats. Furthermore, in TUDCA-treated rat retinas, the number of both rod bipolar and horizontal cell bodies, as well as the density of their synaptic terminals in the outer plexiform layer, was greater than in control rats. CONCLUSIONS. TUDCA treatment was capable of preserving cone and rod structure and function, together with their contacts with their postsynaptic neurons. The neuroprotective effects of TUDCA make this compound potentially useful for delaying retinal degeneration in RP. (Invest Ophthalmol Vis Sci. 2011;52:4998 –5008) DOI:10.1167/iovs.11-7496

R

etinitis pigmentosa (RP) comprises a heterogeneous group of inherited neurodegenerative retinal disorders characterized by progressive peripheral vision loss and night vision

From the 1Departament of Physiology, Genetics and Microbiology, and 3Institute Ramo ´ n Margalef, University of Alicante, Alicante, Spain; and 2Department of Ophthalmology, University Hospital Lozano Blesa, Zaragoza, Spain. Supported by grants from the Spanish Ministerio de Ciencia e Innocación (BFU2009-07793/BFI), Instituto de Salud Carlos III (RETICS RD07/0062/0012), Organizacio ´ n Nacional de Ciegos de Espan ˜a (ONCE), Fundacio ´ n Lucha contra la Ceguera (FUNDALUCE), and Fundacio ´ n de Investigación Me´dica Mutua Madrilen ˜ a. Submitted for publication March 3, 2011; revised April 1, 2011; accepted April 8, 2011. Disclosure: L. Ferna´ndez-Sa´nchez, None; P. Lax, None; I. Pinilla, None; J. Martín-Nieto, None; N. Cuenca, None Corresponding author: Nicola´s Cuenca, Departamento de Fisiología, Gene´tica y Microbiología, Universidad de Alicante, Campus Universitario San Vicente, E-03080 Alicante, Spain; [email protected].

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difficulties, subsequently leading to central vision impairment. More than 100 different mutations in the rhodopsin-encoding gene (RHO) are associated with RP, together accounting for 30% to 40% of autosomal dominant cases. The P23H mutation in this gene is the most prevalent cause of RP,1 which alone accounts for approximately 12% of autosomal dominant RP cases in the United States.2 Most RP-causing mutations in the RHO gene, including P23H, cause misfolding and retention of rhodopsin in the endoplasmic reticulum of transfected cultured cells.3 These studies also suggest that the mechanism of RP involves a cellular stress response,4 the final common pathway of which is programmed photoreceptor cell death, or apoptosis.5 Thus far, no effective therapy has been found for RP. However, several alternative strategies are being investigated to slow or cure this set of diseases. Gene therapy, encapsulated cells releasing neurotrophic factors, and stem cell transplantation are hopeful future approaches to RP treatment.6 Today, gene therapy for RP has been successfully used in both animal models7–10 and humans.11 However, therapy for P23H rhodopsin-induced RP represents a challenge because of its autosomal dominant nature,12 leaving gene replacement and suppression with iRNA as the only therapies envisioned as candidates to lead to cure in the future. It would thus be interesting to address other potential treatments leading, if not to a cure, at least to a delay in RP progression in the short term. Tauroursodeoxycholic acid (TUDCA) is the major component of bear bile and has been used in Asia for more than 3000 years to treat visual disorders. TUDCA has been shown to exhibit antiapoptotic properties in neurodegenerative diseases, including those affecting the retina.13–16 A reduction of photoreceptor apoptosis has been documented, together with a preservation of the a- and b-waves of electroretinograms (ERG), in Pde6brd10 mice (rd10), and in wild-type mice subjected to light-induced retinal degeneration. TUDCA has also proven useful as an antiapoptotic (preventing targeting of the proapoptotic protein Bax to mitochondria) and a cytoprotective agent in animal models of other neurodegenerative disorders, such as Huntington’s,17 Parkinson’s,18 and Alzheimer’s diseases.19 The aim of this study was to evaluate, by means of functional (ERG) and morphologic (histologic labeling) techniques, the effectiveness of TUDCA as an antiapoptotic agent on homozygous P23H line-3 rats, characterized by relatively slow retinal degeneration. Given that one of the first signs of degeneration in these animals was found in the outer plexiform layer (OPL),20,21 we also evaluated its capacity to prevent the loss of synaptic contacts at this retinal location. A positive assessment of the action of TUDCA in this animal model could lead to its possible preventive use in patients with RP. Investigative Ophthalmology & Visual Science, July 2011, Vol. 52, No. 8 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.

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MATERIALS

b-wave and averaged for different recordings. For both scotopic and photopic intensity-response curves, thresholds were defined as the minimal luminance required to reach the criterion amplitude of 10 ␮V.

AND

METHODS

Animals and TUDCA Administration Homozygous P23H line-3 albino rats, obtained from Matthew LaVail (UCSF School of Medicine; http://www.ucsfeye.net/mlavailRDratmodels. shtml), were used in this study. All animals were bred in a colony at the Universidad de Alicante and were maintained under controlled humidity (60%), temperature (23°C ⫾ 1°C), and photoperiod (12hour light/12-hour dark) conditions. All animals were handled in accordance with current regulations for the use of laboratory animals (National Institutes of Health, Association for Research in Vision and Ophthalmology, and European Directive 86/609/EEC) to minimize animal suffering and numbers used for experiments. TUDCA was purchased from Calbiochem (Gibbstown, NJ) and dissolved in PBS just before administration by using an ultrasonic bath to avoid bubble formation. It was administered to P23H rats at 500 mg/kg (intraperitoneally) once a week from postnatal day (P) 21 to P120, and control animals received the same volume of PBS at the same time points. To adjust the amount of TUDCA administered, their body weight was measured before each drug injection.

ERG Recordings After overnight dark adaptation, animals were prepared for bilateral ERG recording under dim red light. Animals were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) plus xylazine (4 mg/kg) solution and were maintained on a heated pad at 38°C. Pupils were dilated by topical application of 1% tropicamide (Alcon Cusí, Barcelona, Spain). A drop of 0.2% polyacrylic acid carbomer (Viscotears; Novartis, Barcelona, Spain) was instilled on the cornea to prevent dehydration and to allow electrical contact with the recording electrodes. These were DTL fiber electrodes with a silver-coated nylon conductive yarn (X-Static; Sauquoit Industries, Scranton, PA). A 25gauge platinum needle inserted under the scalp between the eyes served as the reference electrode. A gold electrode was placed in the mouth and served as ground. Anesthetized animals were placed on a Faraday cage, and all experiments were performed in absolute darkness. Scotopic flash-induced ERG responses were recorded from both eyes in response to light stimuli produced by a Ganzfeld stimulator. Light stimuli were presented for 10 ms at nine different increasing intensities (ranging from ⫺5.2 to 0 log cd 䡠 s/m2). Three to ten consecutive recordings were averaged for each light presentation. The interval between light flashes was 10 seconds for dim flashes and up to 20 seconds for the highest intensity. Photopic responses were obtained after light adaptation at 10 cd 䡠 s/m2 during 20 minutes, and stimuli were the same as for scotopic conditions. The ERG signals were amplified and band-pass filtered (1–1000 Hz, without notch filtering) using a data acquisition board (DAM50; World Precision Instruments, Aston, UK), with data acquisition at 4 kHz (PowerLab; ADinstruments, Oxfordshire, UK). Recordings were saved on a computer and analyzed off-line. The amplitude of the a-wave was measured from the baseline to the trough of the a-wave, and the results were averaged. The amplitude of the b-wave was measured from the trough of the a-wave to the peak of the

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Retinal Histology Retinal Sections. Animals were killed on administration of a lethal dose of pentobarbital, and their eyes were enucleated, fixed in 4% paraformaldehyde, and sequentially cryoprotected in 15%, 20%, and 30% sucrose. After washing in 0.1 M phosphate buffer pH 7.4 (PB), the cornea, lens, and vitreous body were removed, and the retinas were processed for vertical sections. They were then embedded in OCT and frozen in liquid N2. Sixteen-micrometer-thick sections were obtained at ⫺25°C, mounted on slides (Superfrost Plus; Menzel GmbH & Co. KG, Braunschweig, Germany), and air-dried. Before further use, slides were thawed and washed three times in PB and then treated with blocking solution (10% normal donkey serum in PB plus 0.5% Triton X-100) for 1 hour. TUNEL Labeling. Cryostat sections of the retina were stained (Apop-Tag Plus Fluorescein In Situ Apoptosis Detection kit; Chemicon (Temecula, CA). Six retinal sections (containing the optic nerve and both temporal and nasal ora serratae) from seven TUDCAtreated animals and 10 control rats were visualized under a fluorescence microscope (DM IRE2; Leica, Wetzlar, Germany). Retinal Immunohistochemistry. For objective comparison, retinas from vehicle-treated and TUDCA-treated rats were fully processed in parallel. All primary antibodies used in this work (summarized in Table 1) have been used in several previous studies and are well characterized by us and other authors regarding specific cell type molecular marker.22–32 Sections were subjected to single or double immunostaining overnight at room temperature, with combinations of antibodies to different molecular markers at dilutions in PB containing 0.5% Triton X-100 (Table 1). Subsequently, Alexa Fluor 488 (green)– conjugated anti-rabbit IgG or Alexa Fluor 555 (red)– conjugated anti-mouse IgG donkey secondary antibodies (Molecular Probes, Eugene, OR) was applied at a 1:100 dilution for 1 hour. The sections were finally washed in PB, mounted (Citifluor; Citifluor Ltd., London, UK), and coverslipped for viewing under a laserscanning confocal microscope (TCS SP2; Leica). Immunohistochemical controls were performed by omission of either the primary or the secondary antibodies. Final images from control and experimental subjects were processed in parallel using image editing software (Photoshop 10; Adobe, San Jose, CA). Retinal Layer Thickness. Seven TUDCA-treated and 10 untreated animals were examined for measurements of the outer nuclear layer (ONL) thickness. To do that, a nuclear stain (TO-PRO-3 iodide; Molecular Probes) was added at a 1:1000 dilution in at least two sections from each animal containing the optic nerve and both the temporal and the nasal ora serratae. Counting of photoreceptor rows was performed at distances of 0.5, 1.5, 2.5, and 3.5 mm from the optic nerve toward each ora serrata.

TABLE 1. Primary Antibodies Molecular Marker

Antibody

Source

Calbindin D-28K Cytochrome C oxidase, subunit IV Protein kinase C, alpha isoform Bassoon Recoverin

Rabbit polyclonal Mouse monoclonal, clone 20E8 Rabbit polyclonal Mouse monoclonal Mouse monoclonal

Synaptophysin Transducin, G␣c subunit

Mouse, clone SY38 Rabbit polyclonal

Swant Molecular Probes Santa Cruz Biotechnology Stressgen J.F. McGinnis, University of Oklahoma (Oklahoma City, OK) Chemicon Cytosignal

Working Dilution 1:500 1:1000 1:100 1:1000 1:2000 1:500 1:200

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Statistical Analysis Statistical analysis was performed using statistical and graphical software (SYSTAT; London, UK). ANOVA was used to evaluate the effects of TUDCA on ERG responses. To compare the number of TUNELpositive cells found in any experimental group, the two-tailed Student’s t-test was performed. Normal distributions and homogeneity of variance were found for all the analyzed categories. P ⬍ 0.05 was considered statistically significant. Data were plotted as the average ⫾ SEM.

RESULTS TUDCA Preserves Retinal Responsiveness To evaluate the effect of TUDCA on the functional activity of photoreceptors in the P23H rat, scotopic and photopic flash-

IOVS, July 2011, Vol. 52, No. 8 induced ERG responses were recorded in vehicle- and TUDCAtreated animals. As shown in Figure 1, ERG responsiveness was much less deteriorated in rats treated with TUDCA than in control animals. Under scotopic conditions, the maximum amplitudes recorded for a- and b-waves were higher in TUDCAtreated animals, by 44% and 54%, respectively, compared with values obtained in control animals (ANOVA; P ⬍ 0.05 for scotopic a-waves: n ⫽ 18 and n ⫽ 22, respectively; P ⬍ 0.001 for scotopic b-waves: n ⫽ 16 and n ⫽ 20, respectively) (Figs. 1A, 1C, 1D). Similar differences were observed in the maximum amplitudes of photopic a- and b-waves, by 85% and 77%, respectively (ANOVA; P ⬍ 0.05 for photopic a-waves: n ⫽ 5 and n ⫽ 10, respectively; P ⬍ 0.01 for photopic b-waves: n ⫽ 5 and n ⫽ 6, respectively) (Figs. 1B, 1E, 1F). Thresholds in animals treated with TUDCA compared with control rats were smaller for both scotopic b-waves (⫺5.2 log cd 䡠 s/m2 vs. ⫺4.4

FIGURE 1. ERG responsiveness in vehicle- and TUDCA-treated P23H rats. (A, B) Example of scotopic (A) and photopic (B) ERG traces from a P120 rat treated with vehicle (left) or TUDCA (right). Units on the left represent the luminance of the flashes in log cd 䡠 s/m2. (C, D) Stimulus intensity curves for mixed scotopic a-waves (C) and b-waves (D) from rats administered with TUDCA (circles) or vehicle (squares). Scotopic aand b-wave recorded in TUDCAtreated rats reached higher values than those obtained in the control specimens. (E, F) Intensity response of photopic a-waves (E) and b-waves (F). Photopic a- and b-waves recorded in TUDCA-treated rats reached higher values than in control animals.

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log cd 䡠 s/m2) and photopic b-waves (⫺3.2 log cd 䡠 s/m2 vs. ⫺1.9 log cd 䡠 s/m2) (Figs. 1D, 1F).

TUDCA Preserves Photoreceptor Morphology

TUDCA Slows Photoreceptor Degeneration To assess the protective action of TUDCA on photoreceptors, we counted the number of photoreceptor rows present in the ONL at postnatal day (P)120 using a nuclear dye (TO-PRO-3; Molecular Probes). Figure 2A shows a retinal section of a P23H rat treated with vehicle. Fewer rows of photoreceptor cell bodies could be observed in the ONL than in the retinas of TUDCA-treated P23H animals (Fig. 2B). Because retinal degeneration in control P23H rats was not homogeneous throughout the retina, we studied the effects of TUDCA in different retinal areas, from temporal to nasal. We found that the thickness of the ONL was higher in treated than in control animals in all the examined areas (Fig. 2; P ⬍ 0.001 in temporal-nasal areas, and P ⬍ 0.05 in the nasal-peripheral area; Student’s t-test). TUDCA showed its best neuroprotective effect at the ONL level in the 0.5 mm nasal central area of the retina (Fig. 2C), where 4-month-old untreated P23H rats showed approximately two rows of photoreceptor cell bodies (1.9 ⫾ 0.5; Fig. 2A), whereas treated animals showed four to five remaining photoreceptor rows (4.5 ⫾ 0.18; Fig. 2B). Retinal sections from P120 wildtype rats (SD) compared with retinas of TUDCA-treated animals are shown in Supplementary Figs. S1–S4, http://www.iovs.org/ lookup/suppl/doi:10.1167/iovs.11-7496/-/DCSupplemental. To quantify the degree of photoreceptor cell apoptosis in TUDCA-treated and control P23H rats, TUNEL labeling was performed in retinal sections at P120. Figure 3 shows TUNELpositive nuclei located in the ONL. The number of TUNELpositive cells per retinal section was significantly higher (P ⬍ 0.001, Student’s t-test) in control P23H rats (27 ⫾ 10; Figs. 3A, 3C) than in TUDCA-treated rats (11 ⫾ 6; Figs. 3B, 3C).

FIGURE 2. Number of photoreceptor rows in vehicle- and TUDCAtreated animals. (A, B) Retinal sections from P23H rats at P120 administered with vehicle (A) or TUDCA (B), stained with a nuclear marker to visualize the ONL. Both images correspond to the nasal area, 0.5 mm away from the optic nerve. (C) Quantification of the number of rows at the ONL along sections of central retina from the temporal ora serrata to the nasal ora serrata in vehicle- and TUDCA-administered animals (n ⫽ 10 and n ⫽ 7, respectively). *P ⬍ 0.05, **P ⬍ 0.01; Student’s t-test. Scale bar, 10 ␮m.

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To evaluate whether TUDCA was able to preserve the morphology of photoreceptors, we used antibodies against ␥-transducin, a specific marker for cones,28,29 and recoverin, a marker for rods, cones, and two bipolar cell subtypes. In TUDCAtreated P23H rats, rods showed longer inner and outer segments than those observed in vehicle-treated animals (Figs. 4A, 4B), where rod degeneration was more pronounced. Cone photoreceptors underwent even more dramatic changes with age in vehicle-treated P23H rats. At P120 they showed a very small size, with short and swollen outer segments (Figs. 4A, 4C, 4E). Furthermore, axons were absent, and pedicles emerged directly from the cone cell bodies. In contrast, outer segments, axons, and pedicles (Fig. 4F, arrows) and the typical cone shape were preserved in TUDCA-treated animals (Figs. 4B, 4D, 4F). Additional images of sections are shown in Supplementary Figure S1, http://www.iovs.org/lookup/suppl/doi:10.1167/ iovs.11-7496/-/DCSupplemental.

TUDCA Preserves Synaptic Connectivity in the Outer Plexiform Layer We next explored in detail whether the conservation of photoreceptor morphology was accompanied by a preservation of synaptic connectivity in the OPL. To this end, we used antibodies against bassoon, a protein constituent of synaptic ribbons present in both rod spherules and cone pedicles in the OPL. The typical bassoon-immunoreactive spots could be observed, with the horseshoe morphology corresponding to rod spherules (Fig. 5, arrows). Few bassoon-immunopositive spots were found at the OPL level at P120 in P23H rats (Figs. 5A, 5C, 5E, 5G), indicating a decreased number of photoreceptor axon terminals. However, TUDCA-treated animals showed more bassoon-immunoreactive puncta (Figs. 5B, 5D, 5F, 5H), indicating that the presynaptic contact elements between photoreceptors

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FIGURE 3. Photoreceptor cell apoptosis in the P23H rat retina. (A, B) Representative TUNEL immunolabeling of retinas from P120 vehicleadministered (A) and TUDCA-administered rats (B). (C) Graphic representation of the number of TUNEL-positive cells along retinal sections. Note the lower number of labeled cells in TUDCA-treated animals (n ⫽ 7) compared with those observed in the vehicle-treated rats (n ⫽ 10). **P ⬍ 0.01; Student’s ttest. Scale bar, 20 ␮m.

and bipolar or horizontal cells were at least partially preserved. Additional images of sections are shown in supplementary materials (Supplementary Fig. S2, http://www.iovs.org/lookup/suppl/ doi:10.1167/iovs.11-7496/-/DCSupplemental). Double immunolabeling with antibodies against bassoon and ␥-transducin allowed us to identify synaptic ribbons within cone axon terminals. In treated animals, cones showed the typical triangle shape of the axon terminals (Fig. 4F, arrows) compared with the disorganized cone terminal morphology in untreated animals (Fig. 4E). The close relation between synaptic ribbons identified by bassoon immunoreactivity and cone pedicles could be observed in TUDCA-treated animals (Fig. 4F, arrows). However, in untreated animals, cone pedicles were disrupted, and unclear bassoon-immunoreactive spots were found (Fig. 4E).

TUDCA Prevents Loss of ON Bipolar Cell Dendrites and Their Synaptic Contacts with Photoreceptors In the rat retina, dendritic terminals of ON rod bipolar cells established connections with rod spherules through a large dendritic arbor in the OPL, and their axons ran into the IPL, each one ending in a bulbous axon terminal in the S5 stratum. All rod bipolar cells and a particular subtype of amacrine cells were labeled with antibodies against protein kinase C (PKC), ␣ isoform. In the retinas of vehicle-treated P23H rats, rod bipolar cells at P120 showed a substantial loss of cell bodies and a retraction of their dendrites (Figs. 5A, 5C). Dendritic branches were scarce, and some cells were almost devoid of dendrites. The number of immunopositive cells appeared to decrease, and their cell bodies were not aligned in the orderly fashion found in wild-type rats.20 By contrast, in P23H TUDCA-treated animals, bipolar cell dendrites were preserved, and the loss of cells bodies was not so extensive (Figs. 5B, 5D). Double immunostaining for bassoon and PKC showed the relationship between rod photoreceptor axon terminals and bipolar cell dendritic tips. In retinas from vehicle-treated P23H rats labeled at P120 with antibodies against these two markers, few bassoon-positive dots (Figs. 5A, 5C, red) could be seen paired with PKC-labeled bipolar cell dendrites (green). However, in

TUDCA-treated retinas, the number of bassoon-immunoreactive spots associated with bipolar cell dendritic tips was clearly higher (Figs. 5B, 5D).

TUDCA Prevents Loss of Horizontal Cell Dendrites and Their Synaptic Contacts with Photoreceptors Horizontal cell bodies are located in the outermost inner nuclear layer (INL) of the retina and establish connections with both rod and cone photoreceptors. The only horizontal cell subtype described in the rat retina can be identified using antibodies against calbindin. In wild-type rats, calbindin labeling revealed a punctate staining of dendritic arborization protruding from horizontal cell bodies and connecting with cone axon terminals, together with thin tangential axonal elongations in the OPL, ending in an extensive arborization connecting with rods.20 In vehicle-treated P23H rats at P120, a retraction and loss of horizontal cell dendritic tips was found concomitantly with the decrease of TO-PRO-3–stained photoreceptor rows (Figs. 5E, 5G). In TUDCA-treated rat retinas, more horizontal cell terminals could be observed instead (Figs. 5F, 5H). Double labeling with antibodies against bassoon and calbindin showed numerous pairings between photoreceptor axons and horizontal cell terminals in TUDCA-treated animals (Figs. 5F, 5H, arrows) compared with fewer contacts observed in vehicle-treated rats (Figs. 5E, 5G, arrows). This was indicative of a preserving effect of TUDCA on synaptic contacts between photoreceptors and horizontal cells. Additional images of sections are shown in Supplementary Figure S3, http:// www.iovs.org/lookup/suppl/doi:10.1167/iovs.11-7496/-/ DCSupplemental.

TUDCA Preserves Photoreceptor Axon Terminals and Their Synaptic Contacts with Bipolar and Horizontal Cells Given that synaptic ribbons at photoreceptor axon terminals were preserved in TUDCA-treated rats, we tested whether photoreceptor presynaptic terminals were preserved by TUDCA treatment. Toward this end, we performed staining for

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FIGURE 4. Photoreceptor cells morphology in vehicle- and TUDCAtreated animals. Vertical sections of retinas from P23H rats at P120 treated with vehicle (A, C, E) or TUDCA (B, D, F). Nuclei stained with a nuclear marker (blue). (A, B) ␥-Transducin–stained (green) and recoverin-stained (red) retinas showing a more profuse degeneration in the vehicle-treated rat (A) than that observed in the TUDCA-treated rat (B). (C, D) Cone-specific staining for ␥-transducin shows smaller cell sizes and shorter, swollen outer segments in vehicle-treated animals (C) than in TUDCA-treated rats (D). (E, F) Retinas stained for ␥-transducin (green) and bassoon (red) show a lower number of synaptic ribbons in pedicles of vehicle-treated rats (E) than of TUDCA-treated rats (F). OS, outer segment; IS, inner segment. Scale bar, 10 ␮m.

synaptophysin (SYP), a presynaptic-vesicle marker present throughout the axon terminals of cones and rods.29,33,34 In 4-month-old control P23H rats, only an isolated immunoreactive punctate for SYP staining was found, indicating loss of photoreceptor axon terminals giving a discontinuous plexus in the OPL appearance (Figs. 6A, 6C, 6E). In TUDCA-treated P23H rats, a continuous strip of labeled photoreceptor terminals could be observed (Figs. 6B, 6D, 6F), indicating photoreceptor terminal preservation. Double staining for SYP and PKC revealed synaptic contacts between rod spherules (labeled for SYP) and the dendritic tips of rod bipolar cells (labeled for PKC) of TUDCA-treated animals (Fig. 6B, arrows). In comparison, fewer such contacts were observed in vehicle-treated P23H rats (Fig. 6A, arrows). Double staining for SYP and calbindin showed a close relationship between photoreceptor axonal endings and horizontal cell terminals in both control (Figs. 6C, 6E, arrows) and TUDCAtreated (Figs. 6D, 6F, arrows) animals. Rod spherules (labeled for SYP) were associated with the dendritic tips of horizontal cells (labeled for calbindin). However, in the retinas of vehicleinjected animals there was a great loss of photoreceptor axon terminals contacting with the few remaining horizontal cell dendritic tips (Figs. 6C, 6E) compared with those observed in TUDCA-treated animals (Figs. 6D, 6F). High-magnification images of retinas from TUDCA-treated animals labeled for SYP showed well-defined photoreceptor axonal endings, where the empty hole corresponding to their giant mitochondrion was evident (Fig. 6F, arrowheads). Dou-

ble labeling for calbindin and SYP revealed two invaginations for each rod spherule corresponding to a two horizontal cell processes (Fig. 6F, arrows), which was in accordance with the typical morphology of triads formed by axon terminals. These structures were not recognizable in vehicle-treated animals, which indicates a loss of giant mitochondria and the disruption of triads in these animals (Fig. 6E).

TUDCA Slows the Loss of Mitochondria in Photoreceptors To evaluate whether the loss of photoreceptor mitochondria during degeneration was preserved by TUDCA treatment, we examined the staining pattern yielded by antibodies to the subunit IV of cytochrome c oxidase (COX), a molecular marker for the mitochondrial inner membrane system.27 In vehicletreated 4-month-old P23H rats, a narrow strip of COX immunolabeling could be observed in the inner segments of cones and rods (Fig. 7A) compared with the wide strip found in the inner segments of TUDCA-treated animals (Fig. 7B). This result suggested that photoreceptors were healthier in TUDCAtreated animals. At the OPL level, a continuous pattern of COX-immunoreactive spots was found in TUDCA-treated animals (Fig. 7B, arrows), each corresponding to a single giant mitochondrion located in a rod spherule. In vehicle-treated animals, we found a few small, isolated mitochondria, reflecting mitochondrial disruption taking place in the P23H rat at this age. The lack of mitochondria in rod axon terminals agrees

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FIGURE 5. Synaptic connectivity of photoreceptor cells after TUDCA treatment. Immunolabeling of retinal vertical sections at P120 from P23H rats treated with vehicle (A, C, E, G) or TUDCA (B, D, F, H). Nuclei stained with nuclear marker (blue). (A, B) Double immunolabeling for PKC (green) and bassoon (red) showing the preservation by TUDCA of contacts between photoreceptors and bipolar cells (arrows). (C, D) High magnification of A and B, respectively, showing the bassoon-immunoreactive puncta (arrows). (E, F) Double immunolabeling for calbindin (green) and bassoon (red) showing a high number of synaptic contacts between photoreceptor and horizontal cells in TUDCA-treated rats. (G, H) High magnification of E and F, respectively, showing the bassoon-immunoreactive puncta (arrows). IPL, inner plexiform layer. Scale bar, 10 ␮m.

with the observation of flat spherules on SYP staining. Additional images of sections are shown in Supplementary Figure S4, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.11-7496/-/ DCSupplemental.

DISCUSSION The present study shows that systemic TUDCA treatment is capable of preserving retinal structure and function in homozygous P23H transgenic rats. Another bile constituent, bilirubin, has also been shown to have antioxidant activity,35,36 and elevated bilirubin levels in plasma can be protective in diseases involving oxidative stress.37– 40 Ursodeoxycholic acid (UDCA) and its taurine-conjugated analog, TUDCA, exert clear cytoprotective effects in a wide spectrum of diseases, including ocular degenerative disorders such as lens epithelial cell death with cataract formation and ganglion cell death and in both induced and genetic animal models of photoreceptor degeneration.15 In this work we have analyzed the effects of TUDCA on a rat model of autosomal dominant RP characterized by a slow-pace retinal degeneration. We have focused in our study not only on

photoreceptor morphology and function, but also on its secondary effects on photoreceptor connectivity and the structure of inner retinal cell layers. Transgenic P23H albino rats have been engineered to mimic one of the rhodopsin mutations most commonly occurring in human populations.1,2 These rats develop a progressive rod dysfunction, albeit initially exhibiting a normal cone function, which is consistent in broad terms with the clinical findings reported for human patients with P23H RP.21,41 In this animal model, the loss of photoreceptors is accompanied by degenerative changes in the inner retina,20 including a substantial degeneration of retinal ganglion cells.42,43 Even P23H line 1 rats, which undergo retinal derangement at comparatively faster rates than line 3, retain vision for relatively long periods of their lives, similarly to findings in P23H humans, who exhibit significantly better visual acuity and greater ERG amplitudes than patients harboring other RP mutations.41,44 The slow retinal degeneration that takes place in P23H line 3 rats makes this animal model closer to the human condition than other P23H lines and genetic mouse models, thus giving our results additional clinical relevance. In our experiments,

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FIGURE 6. Photoreceptor presynaptic terminals in vehicle- and TUDCAtreated P23H rats. Vertical sections of retinas from P120 rats treated with vehicle (A, C, E) or TUDCA (B, D, F). Nuclei stained with nuclear marker (blue). (A, B) Double immunolabeling for PKC (green) and synaptophysin (SYP; red) showing the preservation by TUDCA of photoreceptor terminals (arrows). (C, D) Calbindin (green) and SYP (red) labeling of retinas showing a high number of photoreceptor axon terminals in contact with horizontal cells in the TUDCAtreated rat. (E, F) High magnification of C and D, respectively, showing photoreceptor axon terminals (arrows) and horizontal cell dendritic tips. Arrowheads: low immunofluorescence in rod terminals corresponding to the space occupied by giant mitochondria. Scale bar, 10 ␮m.

TUDCA was administered from P21 to P120, when vehicletreated animals can be considered to have undergone extensive retinal degeneration (http://www.ucsfeye.net/ mlavailRDratmodels.shtml).45 TUDCA therapy in P23H rats ameliorated the loss of both rods and cones in these animals and preserved their morphology, as evidenced by specific immunostaining of both photoreceptor cell types. Their preservation was in concordance with the higher amplitudes of both scotopic and photopic aand b-waves found in TUDCA-treated animals compared with control animals. Both cone and rod structure and function were preserved to similar extents, as evidenced by the analogous effects found on scotopic and photopic ERG recordings. All these results agree with previous studies carried out in rd10 mice,13–15 an animal model of autosomal recessive RP characterized by rapid rod degeneration followed by cone loss.46,47 TUDCA injections to rd10 mice preserved both rod and cone function and greatly sustained photoreceptor numbers.13–15

FIGURE 7. Mitochondria immunolabeling in vehicle- and TUDCA-treated P23H rats. (A, B) Vertical sections of rat retina stained with anti-COX showing mitochondrion at the OPL and IS levels in a vehicle-treated (A) and a TUDCA-treated (B) rat. Nuclei stained with nuclear marker (blue). (C, D) High magnification of A and B, respectively, showing rod giant mitochondria at the OPL level (arrows). IS, inner segment. Scale bar, 10 ␮m.

However, this positive effect was not always obtained on both photoreceptor types; in another study carried out on rd10 mice, bilirubin and TUDCA were able to preserve cone function and structure, but their effect on rods was only modest and transient.16 This preferential effect on cone photoreceptors has been shown in other animal models of RP using other antioxidative treatments.48,49 In addition to the preventive effects of TUDCA on photoreceptor number, morphology, and function, P23H TUDCAtreated rats experienced improved connectivity between photoreceptors and their postsynaptic neurons: horizontal and bipolar cells. Both presynaptic and postsynaptic elements, as well as synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in TUDCA-treated P23H rats. Furthermore, in the latter, the number of both rod bipolar and horizontal cell bodies, as well as the density of their dendritic terminals, was higher than in vehicle-treated rats. These results strongly indicate that the TUDCA effect on retinal

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morphology and function is not cell specific and, therefore, extends not only to photoreceptors but also to other cell types in the retina. Another interesting possibility is that preservation of the photoreceptor population prevents the occurrence of secondary degenerative changes in their postsynaptic neurons and subsequent retinal circuitry remodeling. It has been proposed that TUDCA effects are exerted through the stimulation of proapoptotic pathways and the endoplasmic reticulum stress response.13–15,50 In our experiments, retinal sections from TUDCA-treated P23H rats showed significantly reduced TUNEL labeling, suggesting that this treatment resulted in at least partial suppression of apoptosis. These results agree with previous experiments carried out in rd10 and light-induced retinal degeneration mice, two animal models in which TUDCA injections prevented photoreceptor apoptosis and ameliorated retinal levels of activated caspase-3.13–15 Antiapoptotic effects of TUDCA have also been demonstrated on other animal models of neurodegeneration, including Huntington’s,17 Parkinson’s,18 and Alzheimer’s diseases.19 Certain apoptotic stimuli are transduced to mitochondria, resulting in the formation of pores and the subsequent release of intermembrane space proteins or the destabilization of the mitochondrial outer membrane.51 The retina, and photoreceptors in particular, is one of the tissues with the highest rates of mitochondrial oxygen consumption and energy demand. In this context, ellipsoids of photoreceptors exhibit a high density of mitochondria that provide energy for phototransduction and the maintenance of Ca2⫹ homeostasis.52 The energy required for synaptic vesicular trafficking and regulation of the cytosolic Ca2⫹ levels in the presynaptic terminals of photoreceptors is provided by a single giant mitochondrion located in each rod spherule and by an average of five medium-sized mitochondria in each cone pedicle.27 Dysfunctional mitochondria cause an energy deficit, leading to an increase of reactive oxygen species (ROS) levels, the activation of mitochondriadependent apoptotic pathways,53,54 and an abnormal elevation of cytosolic Ca2⫹.55,56 Previous works have demonstrated that TUDCA is able to modulate apoptosis by suppressing mitochondrial membrane perturbation.57 In our experiments immunochemistry for COX, the terminal enzyme complex of the mitochondrial electron transport chain located in the inner mitochondrial membrane, revealed a higher number of immunoreactive spots in TUDCA-treated animals than in control rats. A close relationship has been demonstrated between the intensity of COX staining and physiological activity and oxidative metabolism.27,58 Thus, the higher COX staining observed on TUDCA-treated rats compared with untreated rats suggested that this compound reduces mitochondrial damage in the retina and preserves, at least in part, the high rate of energy production required by this tissue. Preservation by TUDCA of mitochondria in presynaptic terminals could be associated with the improvement we found in synaptic connectivity in the OPL of TUDCA-treated rats. Additionally, preservation by TUDCA of mitochondrial function and energy production may contribute to reduce ROS levels in the retina and to prevent the activation of mitochondria-dependent apoptotic pathways, as previously reported in rd10 mice.16 No effective therapy is available to halt the progression of RP or to restore vision once lost. Therapeutic approaches under research thus focus on to how to slow down the degeneration process once started.59 To date, several pharmacologic treatments have been tested on animal models of RP, including vitamins A and E60 and NAD analogs.61 An optimized antioxidant formulation could provide benefit to RP patients by reducing cell death mediated by oxidative stress. Gene therapy for RP has been also successfully used in both animal models7–10 and humans.11 However, although in all cases a significant rescue of photoreceptors was documented, photorecep-

IOVS, July 2011, Vol. 52, No. 8 tor cell death was ongoing, which could have been due to inappropriate expression levels of the therapeutic gene or to an insufficient fraction of photoreceptors becoming transduced. Experiments have also been attempted to transplant retinal cells into the damaged retinas of humans and animal models.6 Stem cells have demonstrated a capacity to regenerate lost photoreceptors and retinal neurons and to improve vision.62 Finally, neuroprotection of the retina has been tested on the delivery of both neuronal growth factors (e.g., ciliary neurotrophic factor)63 and antiapoptotic agents (e.g., proinsulin, melatonin, and TUDCA).13,45,64,65 Despite the use of therapies aimed at preventing cell death, the loss of photoreceptors in number and function usually leads to a dramatic remodeling of retinal circuits that would probably further compromise the transmission of visual information.20 In this context, the use of therapies such as TUDCA, effective not only in preserving photoreceptors from loss but also in slowing the degeneration of inner retinal layers, may be especially interesting in combination with other therapies based on the implantation of new photoreceptors and anti-inflammatory agents, among others.

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SUPPLEMENTARY FIGURE S1. -transducin (green) and recoverin (red) stained retinas from SD (A), vehicle-administered P23H (B) and TUDCA-treated P23H rats (C). Nuclei stained with TO-PRO (blue). OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer. Scale bar: 10 m.

SUPPLEMENTARY FIGURE S2. Double immunolabeling for PKC (green) and bassoon (red) from SD (A), vehicle-administered P23H (B) and TUDCA-treated P23H rats (C). Nuclei stained with TO-PRO (blue). OPL: outer plexiform layer; INL: inner nuclear layer. Scale bar: 10 m.

SUPPLEMENTARY FIGURE S3. Double immunolabeling for calbindin (green) and bassoon (red) from SD (A), vehicle-administered P23H (B) and TUDCA-treated P23H rats (C). Nuclei stained with TO-PRO (blue). ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer. Scale bar: 10 m.

SUPPLEMENTARY FIGURE S4. Vertical sections of rat retina stained with anti-COX from SD (A), vehicle-administered P23H (B) and TUDCA-treated P23H rats (C). Nuclei stained with TO-PRO (blue). IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; Scale bar: 10 m.

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