RhoA and Its Role in Synaptic Structural Plasticity of Isolated Salamander Photoreceptors

Abstract

purpose. Adult salamander photoreceptors retract existing axons and extend new neuritic processes in vitro. In mammalian retina, similar forms of structural plasticity occur in injury and disease. The authors asked whether RhoA is present in photoreceptor axon terminals and whether activity in the RhoA-ROCK pathway contributes to the structural plasticity observed in rod and cone cells.

methods. Antibodies against RhoA were used to immunolabel Western blots sections and isolated neurons from salamander retina. Isolated photoreceptors were treated with lysophosphatidic acid (LPA; a RhoA activator) or Y27632 (an inhibitor of RhoA effector ROCK) for the first 24 hours, the first 3 days, or the last 24 hours of culture. Growth and retraction were assessed with time-lapse and image analyses.

results. RhoA protein was found throughout the retina, including in rod and cone synaptic terminals. When treated with LPA, photoreceptors significantly reduced the growth of new neuritic processes and presynaptic varicosities and retracted growth at the highest LPA concentrations. When treated with Y27632, rod cells significantly increased the number of varicosities, whereas cone cells increased process growth. Treatment with Y27632 also dramatically reduced retraction of the existing axon, which occurs spontaneously in rod cells during the first 24 hours of culture.

conclusions. Thus, RhoA-ROCK activity reduces and retracts neuritic growth, but inhibition of activity increases neuritic development and blocks retraction. The results suggest that RhoA activation contributes to axon retraction by rod cells after retinal detachment, whereas inhibition of RhoA contributes to the neuritic sprouting seen in reattached and degenerating retina.

The synaptic terminals of photoreceptor cells respond to disease and trauma with structural plasticity. Rod and cone photoreceptors produce neurites with synaptic varicosities in degenerative diseases of the outer retina, such as retinitis pigmentosa, ^{1 2} whereas in retinal detachment rod photoreceptor cells retract their axonal fibers and synaptic terminals from the outer synaptic layer. ³ Rod cells have also been observed to sprout new neurites after reattachment. ^{4 5 6} Progress in identifying key signaling molecules responsible for this plasticity has come from examining retraction and growth in isolated salamander photoreceptors and in pig retina maintained in vitro. For instance, blockage of calcium channels and increases in cAMP prevent axon retraction in salamander and porcine rod cells, respectively. ^{7 8 9} On the other hand, increases in cyclic nucleotides stimulate rod and cone cell neuritic growth and synaptic development (Townes-Anderson E, et al. IOVS 2003;44:ARVO E-Abstract 2847). ⁸ Little is known about the signaling pathways these molecules activate in photoreceptor cells. Here we examined the RhoA signaling pathway, which can be modulated by cAMP and cGMP through phosphorylation. ^{10 11 12}  

RhoA, a member of the Rho family of GTPases, is involved in the retraction of axons during development ^{13 14} and the inhibition of new growth after injury. ^{15 16 17 18 19} The Rho GTPases are molecular switches that cycle between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound forms. Once RhoA is activated, it in turn activates downstream effectors, Rho kinase (ROCK), mDia, Rhotekin, protein kinase N, Citron, and collapsing response mediator protein (CRPM4). ^{20 21 22 23} ROCK, the RhoA kinase, phosphorylates various substrates including myosin light chain (MLC), ^{24 25 26 27} LIM kinase, ^{28 29 30 31} Profilin II, ³² and CRMP1 and CRMP2, ^{33 34} all of which have effects on cytoskeleton. 

At present, the activity in the RhoA-ROCK-MLC pathway is perhaps best understood. It is thought to be a key pathway in axon retraction. ROCK phosphorylates both the myosinbinding site of MLC phosphatase, which inhibits the dephosphorylation of MLC, and MLC. ^{35 36} Through these two actions, ROCK increases MLC phosphorylation, leading to actomyosin contraction and presumably to retraction. ^{36 37}  

Inhibiting RhoA/ROCK activity produced a positive effect on growth. Cultured neurons that were prevented from producing new growth by Nogo, MAG, and oligodendrocyte-myelin glycoproteins ^{38 39} were disinhibited when the RhoA pathway was blocked. ^{39 40 41} Similar effects were produced in experimentally induced spinal cord injury. In animal models, RhoA protein and activity have been reported to increase after spinal cord injury. ⁴² In untreated injuries, minimal outgrowth from the area of injury is observed. When RhoA was inhibited, outgrowth increased, and growth was observed beyond the scar created by the injury. ^{16 18}  

In this study, we tested the hypothesis that, in photoreceptors, active RhoA stimulates the retraction of axon terminals and reduces injury-induced neuritic growth, whereas the reduction of RhoA activity stimulates growth. When we treated cultured photoreceptors with a RhoA activator, lysophosphatidic acid (LPA), the number of neuritic processes and varicosities was significantly reduced. In contrast, blocking the RhoA downstream effector ROCK with Y27632 reduced rod axon retraction and increased cone neuritic growth. These results suggest that RhoA plays a role in the structural plasticity observed in the outer retina. Preliminary results from this work have been presented in abstract form (Fontainhas AM, et al. IOVS 2004;45:ARVO E-Abstract 3646). ⁴³  

Methods

Animals

Retinal tissue and cells were obtained from adult, aquatic-phase tiger salamanders (Ambystoma tigrinum, 18–23 cm in length) maintained at 5°C on a 12-hour light/12-hour dark cycle. Animals were adapted to the light cycle for at least 1 week before use. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Medicine and Dentistry of New Jersey and were in strict compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Pharmacologic Reagents

Y27632 (Calbiochem, EMD Biosciences, La Jolla, CA) was dissolved in distilled water, and LPA (Sigma, St. Louis, MO) was dissolved in 0.25% essentially fatty acid-free BSA (Sigma) before they were added to the culture medium. 

Cell Culture

Retinal dissociation was performed as previously described. ^{44 45 46} Animals were decapitated, pithed, and enucleated. The neural retina was placed in a Ringer solution containing papain for 45 minutes. After rinsing and trituration, the cell suspension was plated onto glass coverslips coated with Sal-1 antibody (provided by Peter MacLeish, Morehouse School of Medicine, Atlanta, GA). ⁴⁷ Cultures were maintained in a dark, humidified incubator at 10°C in serum-free medium containing 108 mM NaCl, 2.5 mM KCl, 2 mM HEPES, 1 mM NaHCO₃, 1.8 mM CaCl₂, 0.5 mM NaH₂PO₄, 1 mM NaHCO₃, 24 mM glucose, 0.5 mM MgCl₂, 1 mM Na pyruvate, 7% medium 199, 1× minimum essential (MEM) vitamin mix, 0.1× MEM essential amino acids, 0.1× MEM nonessential amino acids, 2 mM glutamine, 2 μg/mL bovine insulin, 1 μg/mL transferrin, 5 mM taurine, 0.8 μg/mL thyroxin, 10 μg/mL gentamicin, and 1 mg/mL bovine serum albumin (pH 7.7). Cultures were fixed for a minimum of 2 hours with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. 

Retinal Sections

For intact retina, whole eyes were placed in 4% paraformaldehyde for 30 minutes. After the anterior segment was removed, the eyecups were returned to fixative solution and stored overnight at 4°C. Eyecups were rinsed with 5% sucrose, placed in 30% (wt/vol) sucrose/0.1 M PB, embedded in optimum cutting temperature (OCT; Tissue-Tek, Miles Inc., Elkhart, IN) compound, and sectioned with a cryostat (Leica, Allendale, NJ) at 20 μm. The sections were transferred to gelatin-coated slides and stored at −20°C. 

Western Blots

Neural retinas were homogenized and lysed for 15 minutes in ice-cold RIPA buffer (rapid immunoprecipitating assay; adapted from Ren et al. ⁴⁸ ) composed of 50 mM Tris base, 150 mM NaCl, 10 mM MgCl₂, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was clarified with centrifugation at 14,000 rpm for 10 minutes at 4°C (16,000g; Eppendorf, Westbury, NY). Protein content was determined with the Bradford protein assay (Bio-Rad, Hercules, CA). Thirty micrograms of protein, boiled for 4 minutes in 2× Laemmli sample buffer (Sigma), was separated on an 8% to 16% Tris-HCl precast gel (Bio-Rad) at 200 V along with molecular weight markers (Full-Range Molecular Weight Rainbow; Amersham Biosciences, Buckinghamshire, UK). After transfer to nitrocellulose, blots were incubated with a mouse monoclonal (26C4; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal (119; Santa Cruz Biotechnology), or rabbit monoclonal (3L74; Upstate, Charlottesville, NC) RhoA antibody (1:500), followed by appropriate secondary antibodies conjugated to alkaline phosphatase (1:500; Sigma). Staining was developed with Sigma tablets (Fast BCIP/NBT). To determine nonspecific binding, primary antibodies were omitted. For the rabbit polyclonal RhoA antibody, a blocking peptide (sc-117; Santa Cruz Biotechnology) was also used to assess binding specificity. 

Immunocytochemistry

For immunostaining using the avidin-biotin complex (ABC), cells and retinal sections were washed with PBS (450 mM NaCl, 20 mM sodium phosphate buffer, pH 7.4) three times at room temperature and then with 0.5% H₂O₂ in PBS for 10 minutes to inhibit endogenous peroxidase activity, followed by three rinses with PBS. Then cells and retinal sections were incubated for 1 hour at room temperature in 0.1% Triton X-100 goat serum dilution buffer (GSDB; 16% normal goat serum, 450 mM NaCl, 20 mM phosphate buffer) to block nonspecific binding and permeabilize the plasma membrane; primary RhoA antibodies (1:100) were applied overnight at 4°C, followed by three rinses in PBS and one rinse in Triton-free GSDB for 1 hour at room temperature, followed by incubation with biotinylated secondary antibody (1:200) in Triton-free GSDB for 1 hour at room temperature. RhoA antibody was visualized using the avidin-biotinylated horseradish peroxidase (HRP) complex (Vector Laboratories, Inc., Burlingame, CA) developed with stable diaminobenzidine (DAB; Research Genetics, Inc., Huntsville, AL). Labeled sections were dried in 100% ethanol and cleared in Hemo-De (Fisher Scientific, Springfield, NJ), and coverslips were affixed with mounting medium (Permount; Fisher Scientific); labeled cells were stored in PBS. Sections and cells were imaged with brightfield microscopy using an inverted microscope (Axiovert 135; Carl Zeiss, Oberkochen, Germany) equipped with a 40× objective with a 0.75 numerical aperture (N.A.). 

For fluorescence immunolabeling, specimens were treated with a similar protocol without 0.5% H₂O₂. Cells were triple labeled with polyclonal anti-RhoA 119, diluted 1:100, monoclonal anti–rod opsin 4D2 (1:25; generously provided by Robert Molday, University of British Columbia, Vancouver, BC, Canada), and phalloidin conjugated to rhodamine (1:25; Molecular Probes, Eugene, OR) or they were double labeled with anti–RhoA 3L74 (1:100) and 4D2 by the application of antibodies diluted in GSDB plus 0.1% Triton X-100 simultaneously overnight at 4°C. Secondary antibodies conjugated with AF 488, AF 594, and AF 647 (1:35; Alexa Fluor; Molecular Probes) were applied in Triton-free GSDB for 1 hour at room temperature in the dark. Specimens were covered with antifade medium (90% glycerol, 10% PBS, and 2.5% [wt/vol] 1,4-diazobicyclo[2,2,2]octane) to prevent bleaching and were stored in the dark at 4°C. 

Immunolabeling was examined with conventional fluorescence and confocal microscopy. Either a confocal microscope equipped with an argon/krypton laser (LSM410; Carl Zeiss) or a confocal microscope equipped with argon and helium/neon lasers (LSM510; Carl Zeiss)—each with a 63×, 1.4 N.A. oil immersion objective—was used. Specimens were scanned at 1 μm. Cells were examined with an average of 6 to 10 z-sections per cell. 

Time-Lapse Microscopy

To follow axon retraction over time, cultures were viewed with a Zeiss inverted light microscope equipped with a motorized stage controlled by software from Cell Robotics, Inc. (Albuquerque, NM) and a 40×, 0.75 N.A. objective. Twelve to 15 rod cells per dish with axon terminals were selected in the first hour after plating. Each dish was examined by brightfield microscopy every 2 hours for the first 12 hours, and images were captured with a charge-coupled device camera (Sony, Tokyo, Japan). At 24 hours, one last image of each cell was obtained, and dishes were fixed. Axon length was measured with NIH Image software V1.62 (available at rsb.info.nih.gov/nih-image/). 

Analysis of Growth

Photoreceptors were identified by morphology (cell shape, growth pattern, and presence of an ellipsoid), and rod cell identification was confirmed with rod opsin staining. Cells were selected by placing dishes arbitrarily on the microscope stage and then systematically scanning them in rows. Every isolated photoreceptor encountered was digitally captured using Nomarski optics until 25 rod and 25 cone cells per dish were imaged. Neurites 5 μm or larger, originating from the cell body, were counted as main processes. Varicosities 0.5 μm or greater in diameter were counted. Data were collected double blind and were analyzed with one-way ANOVA or χ² tests using commercial software (SigmaStat V3; SPSS Inc., Chicago, IL), and results were plotted (SigmaPlot V8; SPSS Inc.). Data were expressed as mean + SEM. Significance was considered to be achieved at P ≤ 0.05. 

Results

Given that no systematic study has been conducted of the localization of RhoA in amphibian retina, we examined intact retinal sections and isolated retinal cells from salamander with three commonly used antibodies to RhoA. 

Western Blots

The anti–RhoA antibodies 26C4, 119, and 3L74 detected RhoA protein in total salamander retinal protein at around 21 kDa (Fig. 1A) , the reported molecular weight for RhoA. ^{11 49 50} The band frequently appeared as a doublet, with one band greatly reduced in intensity compared with the other. Polyclonal antibody 119 and rabbit monoclonal antibody 3L74 also produced multiple bands at higher molecular weights. Similar patterns for these antibodies have been reported in other tissues. ⁵¹ For polyclonal 119 antibody, peptide-blocking controls significantly reduced all bands except a light 40-kDa band, suggesting that higher molecular weight bands contained some RhoA (data not shown). Collectively, the three antibodies confirmed that RhoA protein is present in salamander retina. Although RhoA is considered the major Rho subfamily protein expressed in tissues, ⁵¹ antibodies 119 and 3L74 also detect RhoB and RhoC. In chick retina, RhoB is downregulated during development and is primarily restricted to Müller cell processes in the inner retina postnatally. ⁵² The presence of RhoC in retina has not yet been directly examined. 

Salamander Retina

To avoid autofluorescence, RhoA was immunolabeled with HRP using the ABC technique in sections of salamander retina. All antibodies tested with Western blot analysis were used. Monoclonal 26C4 anti-RhoA produced a light label throughout the retina (Fig. 1B) , even in the presence of a target unmasking fluid (Signet Laboratories, Inc., Dedham, MA). Polyclonal 119 and rabbit monoclonal 3L74 antibodies produced darker labeling (Figs. 1C 1D) . Nonetheless, all the antibodies yielded a similar labeling pattern. RhoA was present in the ganglion cell layer, inner plexiform layer (IPL), outer plexiform layer (OPL), photoreceptor layers, and Müller cells. 

In photoreceptors, RhoA was present throughout the inner segment, cell body, and axon terminal. It was unclear whether there was labeling in the outer segments because of the presence of the retinal pigmented epithelium. 

Neurons of the INL showed lighter labeling of RhoA compared with the other retinal cells. Müller cell labeling was particularly obvious in the INL because of the light staining of the inner neuronal cell bodies. RhoA labeling of the IPL and ganglion cells was more intense. Fluorescent RhoA labeling of intact salamander retina revealed the same pattern of RhoA localization (data not shown). 

Thus, RhoA was present in normal, uninjured retina, including rod and cone photoreceptors. Because of the consistency of label localization, subsequent labeling was performed with either the polyclonal 119 or the monoclonal 3L74 antibodies, which gave more intense labeling. 

Isolated Retinal Cells

Immunocytochemistry was next applied to isolated salamander retinal cells. Although some rod cells are intact after isolation, most rod cells have lost their outer segments, axon terminals, or both. Rod photoreceptors that retain their axon terminals (also called pedicles) after retinal dissociation normally retract them toward their cell bodies within 24 hours after plating. ⁴⁶ In our cultures, all cone cells lost their outer segments after isolation. Salamander cone cells do not have axons; their synaptic terminals sit on top of the nucleus. Thus, all cone cells retained axon terminals after retinal dissociation. 

Cell isolation stimulates growth in rod and cone photoreceptors. Outgrowth begins within the first 24 hours of culture with the formation of filopodia. After 3 days in culture, photoreceptors produce new neuritic processes with presynaptic varicosities. By 7 days, process growth appears to slow and stabilize. ^{44 53 54} We probed for RhoA within 2 hours after dissociation to determine the normal localization of RhoA and then at 3 and 7 days to determine its localization during periods of growth. 

Brightfield Microscopy.

Using the ABC-labeling technique and polyclonal 119 primary antibody, RhoA was found in all retinal cell types (rod and cone photoreceptors and horizontal, bipolar, amacrine, ganglion, and Müller cells). 

Two hours after dissociation, rod cells contained RhoA labeling throughout the entire cell (Figs. 2A 2C) , including the axon terminals. Labeling was also present in rod pedicles in the process of retracting toward the cell body (Fig. 2A) . With isolated rod cells, it was clear that RhoA labeling was present in the outer segments. Cone cells were also diffusely immunolabeled. Cone pedicles contained RhoA, evidenced by slightly darker staining at the nuclear pole (Fig. 2B) . The highest intensity of RhoA label for rod and cone cells occurred in the inner segments. 

RhoA continued to be present in 3- and 7-day cultures (Figs. 2E 2F 2G 2H) . Immunolabel was present in filopodial and lamellipodial processes and thicker neurites with varicosities. As at 2 hours, RhoA labeling appeared to be prominent in the inner segments. 

Confocal Microscopy.

To determine whether RhoA was present at the plasma membrane of the photoreceptors and what the relative abundance of label was in subcellular regions, fluorescence-labeled photoreceptors were examined in 1-μm optical sections. Cells were triple labeled for RhoA (119), rod opsin and f-actin or double labeled with antibodies against RhoA (3L74) and rod opsin. Labeling with polyclonal 119 and monoclonal 3L74 anti–RhoA antibodies yielded identical labeling patterns. RhoA immunoreactivity was present throughout the cytosol of the cell body, inner segment, ellipsoid, and axon terminal 2 hours after dissociation (Figs. 3A 3a 3B 3b) . Rod and cone pedicles showed an equivalent intensity of RhoA immunolabel. In the inner segments, rod and cone cells had intense accumulations of label. Three days in culture did not appear to affect the levels of RhoA in retinal cells. RhoA was observed in the thick processes and varicosities of rod and cone cells (Figs. 3C 3D) . A clustering of RhoA signal was also found in the inner segment of rod and cone photoreceptor cells (Figs. 3c 3d) . 

RhoA plays a role in maintaining the normal structure of the Golgi apparatus in hippocampal neurons. ²² In rod cells, some RhoA immunolabel is likely to be in the Golgi apparatus because opsin occurs at high levels in the Golgi of cultured photoreceptor cells ⁴⁶ and because RhoA immunolabeling partially colocalized with opsin label at the location of the putative Golgi apparatus (Figs. 4A 4a ′, 4a″; see also Figs. 3C 3D ). In contrast, RhoA did not colocalize with opsin label in the plasma membrane (Fig. 4)or with submembranous actin (Fig. 3) . RhoA also controls mitochondrial movements. ⁵⁵ Staining in the ellipsoid, which is an accumulation of mitochondria, was observed in rod and cone cells with both the ABC and the fluorescence immunolabeling techniques (Figs. 2 3 4) . 

Thus, two types of immunolabel (ABC and fluorescence) that require different microscopic techniques gave consistent results. Both techniques demonstrated that RhoA protein is present in retinal cells throughout the time in culture. RhoA was found in photoreceptor cells as they engaged in structural remodeling, and it was present in axon terminals during retraction and in neuritic processes during growth. 

Photoreceptor Neuritic Growth

To determine how RhoA functions in photoreceptor structural plasticity, drugs were used either to activate RhoA or to block RhoA activity. RhoA was activated by LPA, and RhoA downstream effector ROCK was inhibited with Y27632. 

LPA, RhoA Activator.

Lysophosphatidic acid has been shown to be an activator of the RhoA protein working through receptors associated with the G_α13 protein. ^{56 57} To examine the effects of RhoA activation on established growth, retinal cells were allowed to grow for 2 days. At the end of the 2-day period, LPA was applied at 2, 20, and 200 μM for the next 24 hours. Control cultures were fixed at day 2 or day 3. By comparing average growth in 2- and 3-day control rod and cone cells, it appeared that rod photoreceptors increased processes and varicosities until day 3 (Figs. 5A 5B 5D) , whereas cone cells grew more rapidly, achieving maximal process growth by day 2, though varicosities continued to form (Figs. 5E 5F 5G) . 

Rod photoreceptor cells reduced the number of processes and varicosities in response to LPA compared with control growth at 3 days; reductions in growth increased with higher LPA concentrations (Figs. 5C 5D) . With 20 and 200 μM LPA, varicosities were also significantly reduced in rod cells compared with the 2-day controls. Although the reduction in processes and varicosities for rod cells indicated an inhibition of growth with LPA, reduction in growth compared with 2-day controls indicated some retraction of established growth at the higher LPA concentrations as well. 

Cone photoreceptor cells did not significantly reduce the number of processes with 2 and 20 μM LPA treatment compared with 2- and 3-day controls. However, with 200 μM LPA, cone cell processes were significantly reduced compared with 2- and 3-day controls (Fig. 5E) . Cone cell varicosities responded in a manner similar to that of rod cell varicosities. Cone cell varicosities were significantly reduced in a linear fashion as the LPA concentration increased compared to 2- and 3-day cultures (Figs. 5E 5H) . Thus, cone cell processes retracted in response to the highest LPA concentration, whereas varicosities were affected, and presumably retracted, at all levels of LPA treatment. 

To look at effects of RhoA activation on the initiation, and the maintenance, of growth, LPA was added at 2 and 20 μM to the culture medium for all 3 days of culture. Both concentrations of LPA resulted in a significant decrease of processes and varicosities in rod and cone cells (Figs. 6A 6B) . In contrast to the shorter 24-hour treatment, longer exposure to lower concentrations (2 and 20 μM) of LPA was able to reduce process outgrowth in cone cells. 

Y27632, ROCK Inhibitor.

If RhoA activation through LPA reduces growth, inhibition of RhoA activity by inhibition of the ROCK pathway may have a stimulatory effect on growth. Y27632 is a specific inhibitor of ROCK. ⁵⁸ In preliminary experiments, Y27632 was applied at different concentrations (25, 50, and 100 μM) and different lengths of time, according to the protocol for LPA. The largest effect on growth was observed with the 100-μM concentration present for the full 3 days of culture (data not shown). These results were repeated using 100 μM Y27632 in cultures treated for 3 days (Figs. 6C 6D) . Examination of rod cells showed no significant increase in the growth of processes; however, there was a small but significant increase in the number of varicosities (Fig. 6C) . In contrast, for cone cells, there was a significant increase in processes and varicosities (Fig. 6D) . Thus, blocking ROCK activity had limited effects on rod growth but stimulated cone growth. 

Blocking the LPA Effect with Y27632.

Finally, LPA is capable of signaling through diverse pathways. Depending on the combination of receptors and G-proteins, LPA can activate PLC, MAPK, Akt, PI3K, and RhoA and can inhibit adenylyl cyclase. ⁵⁹ To verify that LPA reduces growth through the activation of the RhoA pathway in photoreceptors, we blocked the downstream effector ROCK with Y27632 before stimulating cells with LPA. When 200 μM LPA was applied to cultures pretreated for 2 hours with 100 μM Y27632, there was no significant decrease (or increase) of main processes and varicosities in rod and cone cells after 3 days in culture, whereas LPA treatment alone, as expected, significantly decreased the number of processes and varicosities (data not shown). These results demonstrated that LPA activated the RhoA-ROCK pathway given that inhibiting ROCK successfully blocked its effect. 

Rod Axon Terminal Retraction

In addition to growth, injury can produce process retraction. Indeed, one of the first observable changes in the structure of rod photoreceptors in culture is the retraction of the rod pedicle. ⁴⁶ RhoA is likely to be involved in this process. Isolated retinal cells were plated into culture dishes containing salamander medium either alone or with 100 μM Y27632 or 200 μM LPA. Rod cells without outer segments but with their axon terminals were selected within the first hour after plating and were examined over a 24-hour period. 

In control cultures, noticeable changes in rod axon terminal shape were observed by 6 hours (Figs. 7A 7B) . After 12 hours in culture, retraction of axons was obvious (Fig. 7C) ; by 24 hours, most axons were fully retracted into the cell body (Fig. 7D) . A similar pattern was evident with LPA treatment (Figs. 7I 7J 7K 7L) . However, this process was different in rod cells treated with Y27632. There was no visible decrease of axon length from 1 hour to 24 hours (Figs. 7E 7F 7G 7H) , and some rod axons actually increased in length by the end of 24 hours (Fig. 7H) . 

In control and LPA-treated cultures, 81% and 85% of the selected rod cells, respectively, retracted their terminals by 24 hours (Fig. 7M) . With the ROCK inhibitor Y27632, only 49.5% of the selected and treated rod cells retracted their axon terminals. Thus, blocking the RhoA effector ROCK with Y27632 had a significant impact on the number of rod axon terminals that retracted in the first 24 hours of culture (Fig. 7M) . 

Changes in the length of the axonal fiber were measured over time (Fig. 7N , inset). The mean rod axon length in control rod cells was reduced by half by 12 hours, and by 24 hours it was significantly shorter than it was at 1 hour. A similar scenario occurred for the LPA-treated group. There was no significant difference in average axon length between LPA and control rod photoreceptors at 24 hours. However, the mean rod axon length of the Y27632-treated group did not significantly decrease over time. Examined collectively, average axon length at 1 hour for all three groups was not significantly different, whereas at 24 hours there was a significant difference in average length between Y27632 treatment and the control and LPA groups (Fig. 7N) . 

The fact that the number and average length of axons were maintained over a 24-hour period, when ROCK activity was inhibited by Y27632, signified that the RhoA-ROCK pathway, when active, leads to axon terminal retraction. Interestingly, the amount of retraction was not different between control and LPA groups, suggesting that components of the RhoA pathway involved in retraction in rod cells were either maximally activated or refractory to further stimulation during the 24 hours immediately after plating. 

In addition to monitoring axonal retraction, filopodial growth was examined during the 24-hour culture period (Fig. 8A) . For rod cells, LPA significantly reduced the number of filopodia, but Y27632 did not significantly increase filopodia. This was consistent with the effects of LPA and Y27632 on rod cell growth over 3 days. Moreover, it demonstrated that LPA inhibited growth on the first day of culture even though it did not promote retraction beyond control levels. For cone cells, LPA reduced the numbers of processes and varicosities during the first 24 hours of culture, whereas Y27632 increased growth significantly (Fig. 8B) . These results were also consistent with the effects of these treatments on cone cells over 3 days. Therefore, during early and extended times in culture, rod and cone cells responded differently to inhibition of ROCK activity. Blocking ROCK in rod cells affected retraction but not process growth. In cone cells, which do not retract their pedicles, the ROCK inhibitor Y27632 stimulated growth. 

Discussion

RhoA is a multifunctional protein ^{35 36} that plays a role in nerve cell process retraction and growth. Retinal detachment leads to axon terminal retraction from the outer plexiform layer by rod cells. ³ In reattached retina and many forms of retinal degeneration, rod photoreceptors have been observed to sprout neurites into the inner retina. ^{4 5} In the mouse rd1 model of retinal degeneration, cone cells sprout neurites. ⁶⁰ Isolated salamander rod and cone photoreceptor cells maintained in culture produce similar structural plasticity: isolated rod photoreceptors retract axon terminals, and rod and cone cells grow varicosity-bearing neuritic processes over time in culture. We hypothesized that RhoA is involved in axon terminal retraction and growth and examined this question in vitro in isolated and cultured amphibian photoreceptors. 

The presence of RhoA in adult salamander retina was demonstrated using multiple antibodies and immunolabeling techniques. The functional role of RhoA was tested by manipulating RhoA activity and interfering with its effector ROCK. In rod cells, LPA reduced the number of neuritic processes and caused retraction of varicosities at high concentrations, whereas ROCK inhibition had little effect on growth but did block axon retraction. In cone cells, LPA reduced the numbers of processes and varicosities causing retraction of existing growth; conversely, ROCK inhibition increased the number of processes and varicosities compared with control. Our results support the conclusion that active RhoA-ROCK promotes axonal retraction and inhibits neuritic growth and that reduction in active RhoA-ROCK signaling promotes injury-induced growth. The data also suggest that rod and cone cells have differences in RhoA signaling because inhibition of the downstream effector ROCK had different outcomes in rod and cone cells. It appears that ROCK activity stimulates axon retraction in rod cells but inhibits the growth of neuritic processes in cone cells. 

In addition to salamander retina, RhoA has been observed in chick and mouse retina during development and in rat and mouse adult retina. ^{51 61 62} In postnatal chick and adult mouse retina, RhoA was present in inner segments and the OPL. Thus, as in salamander cone and rod photoreceptors, RhoA is present in avian and mammalian cone and rod synaptic terminals, respectively. ^{61 62 63}  

What purpose does RhoA serve in the adult retina? It may play multiple roles in the maintenance of structure. The inhibition of prenylation in the retina by levostatin led to rosette formation in the photoreceptor layer. ⁶⁴ Rho GTPases (including RhoA) are posttranscriptionally modified by prenylation ⁶⁵ of the geranylgeranyl moiety. ^{66 67} GTP- and GDP-bound forms of RhoA are prenylated. Prenylation is necessary to anchor active RhoA to the plasma membrane and to allow binding of GDI, which inhibits RhoA activity. ^{66 68} The effects of levostatin suggest that the Rho GTPases are involved in the maintenance of lamination. RhoA may directly affect adherens junctions, which are found between photoreceptors at the outer limiting membrane. In epithelial tissue, the RhoA-ROCK pathway disrupts adherens junctions, whereas the RhoA-mDia pathway is required for the maintenance of adherens junctions. ⁶⁹ RhoA is also implicated in cadherin clustering necessary for the formation of adherens junctions. ⁷⁰ In the outer segment, RhoA may play a role in signal transduction. RhoA has been found in bovine rod outer segments. ^{71 72} Phospholipase D (PLD), an enzyme found in ROS, is involved in lipid signal transduction and membrane trafficking. ^{73 74} Salvador and Giusto ⁷¹ have linked active RhoA with reduced PLD activity. Taken together, these data support the idea that RhoA helps maintain the normal architecture of the adult retina. 

Our results suggest that RhoA also plays a role in the response by the adult retina to injury and disease. RhoA has been linked to retraction of axons and inhibition of growth after trauma in several parts of the central nervous system (CNS). ^{16 17 24 42 75 76 77} When neurons in the spinal cord, optic nerve (retinal ganglion cells), or brain undergo trauma, RhoA activity is rapidly increased. ^{16 75 78} Adverse effects of active RhoA, in the form of retraction and synaptic loss, can be reduced if RhoA or ROCK activity is blocked with C3 transferase or Y27632, respectively. ^{16 75 78} It is possible that retinal detachment activates RhoA, which induces rod axon terminal retraction using the RhoA-ROCK pathway. After reattachment or in retinal degenerative disease, it is possible that RhoA activity is reduced, allowing photoreceptor sprouting. 

In an in vitro model of retinal detachment, treating detached porcine retina with cAMP reduced the retraction of rod synapses. ⁹ cAMP may do this by inhibiting ROCK activity. PKA phosphorylates RhoA at Ser188. ¹² The phosphorylation of RhoA does not stop RhoA from becoming active, but it does inhibit GTP-bound RhoA (active RhoA) from interacting with the downstream effector ROCK while still interacting with other RhoA effectors, such as protein kinase N (PKN), mDia, and Rhotekin. ⁷⁹  

How is RhoA activated in photoreceptors? Activation of RhoA after injury in the CNS has been linked to inhibitory myelin-derived signals. ^{16 39 40 78} Myelin is not present in the outer retina, where the photoreceptors are located. Direct mechanical stress produced during detachment, or retinal dissociation, may activate RhoA. Mechanical stress has been shown to increase RhoA activity in smooth muscle cells. ⁸⁰ In the intact retina, it is also possible that microglia activate RhoA. ⁸¹ Fisher et al. ⁸² and others ^{83 84} have shown that retinal microglia are activated after detachment. Activated microglia release tumor necrosis factor (TNF), which increases RhoA activity, reducing neuritic length and branching in cultured hippocampal neurons. ⁸¹ RhoA activation in vivo may also be stimulated by LPA. ¹³ LPA is produced by the retinal pigment epithelium, ⁸⁵ and LPA-1 and -2 receptors are found throughout normal rat retina. ⁸⁶ This raises the possibility that during retinal detachment LPA is produced and stimulates RhoA activation through these receptors. Photoreceptors, in salamander at least, appear to have LPA receptors because they respond to LPA stimulation by reducing growth. 

Once activated, the RhoA-ROCK pathway may work differently in rod and cone cells. For rod cells in culture, activity in the RhoA-ROCK pathway was vital for axon terminal retraction but appeared subsequently to be suppressed. Inhibiting ROCK had no positive effect on new neuritic growth. Cone cells, on the other hand, did not seem to downregulate the RhoA-ROCK pathway, and manipulation with Y27632 to reduce ROCK activity led to an increase in growth at all times in culture. It is possible that the levels of active RhoA or the levels of ROCK and its activation are different in the two cell types. Other explanations include differences in other Rho family members, such as Rac1. In the CNS, Rac1 can regulate RhoA activity and vice versa: high levels of Rac1 can block RhoA activity. ^{87 88} Thus, levels of Rac1 may also vary. Alternatively, levels of cyclic nucleotides may vary after injury in a cell-type specific manner. An increase in cyclic nucleotides would have a regulatory impact on the RhoA-ROCK pathway because cAMP and cGMP are able to inhibit RhoA interaction with ROCK. ^{11 12 23} Differences in the mechanisms for structural plasticity in rod and cone cells are not surprising because it has already been reported that the NO-cGMP-PKG pathway has different effects on neuritic sprouting by rod and cone cells. ⁸⁹  

In conclusion, we have shown that RhoA is present in the photoreceptor synapse. In a salamander cell culture system, modulating RhoA contributes to rod axon retraction and to rod and cone neuritic sprouting, structural changes that are also seen in retinal detachment and retinal degeneration. 

 Supported by National Eye Institute Grant EY012031 and by the F.M. Kirby Foundation.

 Submitted for publication December 10, 2007; revised April 3 and May 5, 2008; accepted July 17, 2008.

 Disclosure: A.M. Fontainhas, None; E. Townes-Anderson, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

RhoA in salamander retina. (A) Western blot. Multiple antibodies against RhoA protein label a 21-kDa band (arrow; 26C4, a mouse monoclonal antibody; 119, a rabbit polyclonal antibody; and 3L74, a rabbit monoclonal antibody). (B–D) RhoA, visualized in retinal sections with the ABC technique. Although staining intensity varies, the patterns of monoclonal and polyclonal RhoA immunolabel are similar regardless of the primary antibody. RhoA is present in the inner segment (IS), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), and Müller cells (arrowheads). (E) Control, primary antibody was omitted.

Figure 2.

 

RhoA in photoreceptors. RhoA, immunolabeled with the ABC technique, is found in rod (A, C, E, G) and cone (B, C, F, H) cells 2 hours, 3 days, and 7 days after isolation. (A, C) Rod cells exhibit RhoA throughout the cell, including the outer segment and axon terminal (arrows). Labeling is present as the axon terminal retracts (A, arrow). (B, C) Cone cells show RhoA labeling throughout the cell, including the synaptic pedicle (B, arrow). Darker labeling appears in the inner segments of rod and cone cells (A–C, double arrowheads). Rod (E) and cone (F) photoreceptors produce filopodial processes and neurites bearing varicosities by day 3 in culture. RhoA is dispersed throughout the cell body and its processes and varicosities (arrowheads). After 7 days in culture, rod (G) and cone (H) cells show RhoA labeling throughout the cell body, in all new growth and in the varicosities (arrowheads). (D) Control rod and cone cells, produced by omitting the primary antibody, have no labeling. Arrow indicates rod axon terminal. n, nucleus; e, ellipsoid; os, outer segment. Scale bar, 30 μm.

Figure 3.

 

One-micrometer confocal sections of rod and cone cells after 2 hours (A, B) and 3 days (C, D) in culture. (A–C) Photoreceptors triple labeled for RhoA (green), f-actin (red), and rod opsin (blue). (D) Rod cell double labeled for RhoA (green) and rod opsin (red). (a–d) The green (RhoA) channel is shown monochromatically. (A, a) RhoA is present throughout the rod cytoplasm, including the axon terminal (arrow), and is accumulated in regions of the inner segment (double arrowheads). Opsin is present in the plasma membrane and the inner segment. (B, b) In cone cells, RhoA is present throughout the cytoplasm, with areas of increased intensity in the inner segment (double arrowheads). Labeling basal to the nucleus shows that RhoA is present in the flattened pedicle (arrow). (C, c) RhoA continues to be present at 3 days. The rod cell, labeled for rod opsin, has abundant filopodial growth filled with f-actin. The cone cell has fewer actin-filled processes. RhoA labeling is present throughout the cytoplasm, with the most intense staining in the inner segment (double arrowheads). (D, d) RhoA is also present in the varicosity (arrowhead) of a 3-day rod cell. Inset: control, primary antibody was omitted. Scale bar, 10 μm.

Figure 4.

 

RhoA colocalizes with rod opsin in the inner segment. A 1-μm confocal section of a 3-day cultured rod cell double labeled for RhoA and opsin. (A) RhoA and rod opsin colocalize in the inner segment (yellow) between the nucleus and the ellipsoid. The green channel (a′) shows RhoA label throughout the soma of the cell, with the highest intensity of labeling in the inner segment. The red channel (a″) shows rod opsin localization at the plasma membrane and in the inner segment, with the highest intensity in the presumed Golgi apparatus.

Figure 5.

 

Growth is reduced and retracted in rod and cone cells by 24 hours of LPA treatment after 2 days of growth. (A–C) Representative rod cells from 2- and 3-day control cultures and after 20 μM LPA treatment. Rod cells produce growth over 3 days (varicosity, arrowhead), which is reduced by 20 μM LPA treatment. (D) Rod cell processes and varicosities increased from 2 to 3 days in control cultures. LPA at 2, 20, and 200 μM reduced the number of processes and varicosities compared with 3-day controls. Rod varicosities showed a steady decrease in number as LPA concentration increased. With 20 and 200 μM LPA, varicosities were also significantly reduced compared with 2-day controls. n = 4 animals, 20 culture dishes, 500 rod cells; *P ≤ 0.001. (F–H) Representative cone cells from 2- and 3-day control cultures and after 20 μM LPA treatment. Neuritic growth, including varicosity development (arrowhead), is already present by day 2. It is reduced by 20 μM LPA. (E) The 2- and 3-day control cone cells showed no difference in number of processes. LPA at 200 μM reduced the number of processes below those of the 2- and 3-day controls. Cone varicosities increased from 2 to 3 days in control cultures but showed a linear decrease in number compared to 2- and 3-day controls as LPA concentration increased. n = 4 animals, 20 culture dishes, 500 cone cells; *P ≤ 0.001.

Figure 6.

 

Growth is reduced in rod and cone cells by prolonged 3-day LPA treatment, whereas Y27632 treatment (a ROCK inhibitor) results in increased growth. Rod (A) and cone (B) cells treated with 2 or 20 μM LPA over 3 days in culture reduced the number of processes and varicosities compared with 3-day control cells. n = 4 animals, 12 culture dishes, 300 rod cells, 300 cone cells; *P ≤ 0.001. (C) Rod cells treated with 100 μM Y27632 significantly increased the number of varicosities. n = 6 animals, 12 culture dishes, 300 rod cells; *P ≤ 0.001. (D) Cone cells treated with 100 μM Y27632 increased the number of processes and varicosities. n = 3 animals, 6 culture dishes, 150 cone cells; *P ≤ 0.001.

Figure 7.

 

Retraction of the rod photoreceptor axon terminal is reduced by ROCK inhibition. (A–L) Rod photoreceptors in control and treated cultures, 100 μΜ Y27632, and 200 μM LPA at 1, 6, 12, and 24 hours. Circles highlight the axon terminal. (A–D) Rod cell in a control culture shows axon terminal retraction into the cell soma within 24 hours and no growth from the site of the axon terminal. (E–H) Rod cell treated with Y27632 retains its axon terminal throughout the 24-hour period and produces additional axonal growth. (I–L) Rod cell treated with LPA retracts its axon terminal within 24 hours, similar to control. (M) Inhibiting ROCK, RhoA effector, with Y27632 reduced the number of rod cells that retracted their axons. (N) Inhibiting ROCK, with Y27632, prevented a reduction in the average length of axons. Length was measured from the tip of the axon terminal to the soma, as shown in the inset. Control and LPA-treated rod cells reduced their axon length by 24 hours. No decrease in the mean length of axons over time in Y27632-treated rod cells was observed. n = 4 animals, 24 culture dishes, 242 rod cells; *P ≤ 0.001.

Figure 8.

 

Growth during the first 24 hours of culture is affected by ROCK inhibition in cone but not in rod cells. (A) For rod cells, growth in the form of filopodia was not significantly affected by Y27632 treatment but was significantly reduced with LPA treatment. n = 4 animals, 24 culture dishes, 242 rod cells; *P ≤ 0.001. (B) For cone cells, the number of processes and of varicosities in the first 24 hours of culture increased with Y27632 treatment, whereas LPA treatment decreased processes and varicosities. n = 4 animals, 32 dishes, 480 cone cells; *P ≤ 0.001.

The authors thank Robert Clarke and Youhua Zhu for assistance with data analyses and Emily Brewster for assistance with double-blind experiments.