Light-Induced Photoreceptor Degeneration in the Mouse Involves Activation of the Small GTPase Rac1

Abstract

purpose. Rho GTPases play a central role in actin-based cytoskeleton reorganization, and they participate in signaling pathways that regulate gene transcription, cell cycle entry, and cell survival. This study verifies the role of Rac1 during light-induced retinal degeneration.

methods. BALB/c mice were exposed to degenerative light stimulus, and their eyes were enucleated immediately or after the mice were kept in the dark for 6, 24, and 48 hours. Retinas were fixed and processed for immunohistochemical analysis. The distribution of Rac1 and its effectors—p21-activated kinases (PAKs) 1, 2, and 3—was studied by immunohistochemistry, whereas the expression of PAKs 3, 4, and 5 mRNA was analyzed by real-time PCR. Rac1 activity was measured using a pull-down assay.

results. In control retinas, Rac1 was mostly observed in photoreceptors, plexiform layers, and Müller glial cells. In light-damaged retinas, some TUNEL-positive photoreceptors upregulated Rac1 expression. Conversely, most of the Rac1-positive cells were TUNEL-positive, mainly in early stages of retinal degeneration. The increase in Rac1 expression was preceded by enhanced Rac1 activity, detectable at the end of the light stimulus and still present 48 hours later. The distribution patterns of PAK1, PAK2, and PAK3 did not change in light-damaged retinas. However, there was a marked increase in PAK3 and PAK4 gene expression, whereas that of PAK5 mRNA remained the same.

conclusions. Rac1 may play a role in the apoptosis of light-damaged photoreceptors. The increased expression of PAK4 after light stimulus possibly functions as a protective mechanism against apoptosis.

Most of the inherited retinal degenerative diseases share the common feature of photoreceptor cell death followed by morphologic and functional disruption of the internal retina during the degenerative process. ¹ In patients with retinitis pigmentosa (RP), which afflicts thousands worldwide every year, this degeneration results in a clinical phenotype of progressive night blindness and tunnel vision, eventually culminating in a devastating loss of visual function. ²  

Most genes implicated thus far in the etiology of this disease encode photoreceptor-specific proteins that are structural components of these cells or that participate in the phototransduction cascade. The pathologic mechanism underlying RP has not been determined with certainty, but independent studies in genetic animal models (including the rd1 mouse) and in light-injured experimental animals lead to the conclusion that apoptosis is the final common pathway of photoreceptor death. ^{3 4 5} The elucidation of the retinal pathways and molecules involved in apoptotic death has been helpful for the development of new strategies to prevent RP and other neurodegenerative disorders. ^{6 7 8 9}  

The Rho family of small GTPases has been implicated in the regulation of several cell functions, including a variety of actin-dependent processes such as migration, adhesion, cytokinesis, membrane traffic and axon guidance, cell cycle, differentiation, and apoptosis. ^{10 11 12 13 14} Rho proteins play a role in the signaling from extracellular stimulation to intracellular downstream effectors, cycling between an inactive guanosine diphosphate (GDP)–bound state and an active guanosine triphosphate (GTP)–bound state. ^{12 15} The best known proteins in the family are the widely distributed GTPases RhoA, Rac1, and Cdc42. 

The activity of Rho GTPases is amplified in a variety of signal transduction pathways through its different downstream effectors. ¹⁶ For example, the serine/threonine kinases P21-activated kinases family (PAKs), first identified as Rac1 and Cdc42 effectors, ¹⁷ are important for the regulation of neuronal cell morphology through their effects on the actin cytoskeleton. ^{18 19} With the recent discovery of novel isoforms, PAKs are now categorized into two subgroups according to their structural similarities, namely group 1 (PAK1, PAK2, PAK3) and group 2 (PAK4, PAK5, PAK6). Although group 1 PAKs have been studied in detail and have been shown to be involved in the regulation of cellular processes such as gene transcription, cell morphology, motility, and apoptosis, less is known about the group 2 PAKs. ²⁰  

Several studies have associated Rho signaling to survival and apoptosis events. ^{21 22 23 24 25 26} In the nervous system, both proapoptotic and antiapoptotic roles have been established for these proteins and some of their effectors. ^{27 28 29 30} Interestingly, rescue of degenerating photoreceptors by activated Rac1 was described in rhodopsin-null Drosophila mutants. ³¹ Most effects on cell survival are related to the activation of Akt and PI3-kinase and to the phosphorylation of Bad and other Bcl-2 proteins. ^{32 33} Conversely, the proapoptotic effects are mostly related to the production of reactive oxygen species by Rac1. ^{34 35 36}  

In the chick retina, Rho GTPases are expressed by neurons and Müller cells in the adult and during development. ³⁷ Furthermore, Rac1 is present in mouse photoreceptor segments and is activated by light. ³⁸ Thus, we hypothesized that Rac1 might play a role in normal eye function and that alterations in its expression might be correlated to the cell death process in light-overstimulated photoreceptors. Here we characterize the expression pattern and levels of Rac1 and its effectors—PAKs 1, 2, 3, 4, and 5—in normal mouse retina while comparing it with light-injured retina undergoing degeneration. The unavailability or inconsistency of commercial antibodies against PAK4 and PAK5 lead us to study their mRNA expression through RT-PCR only. Because PAK3 was highly expressed in photoreceptors, we decided to study its distribution and its mRNA expression. 

First, we demonstrated that Rac1 was upregulated only in a subpopulation of dying photoreceptors (TUNEL-positive) during light-induced retinal degeneration. In addition, preceding its expression increase in photoreceptors, Rac1 activity in the retina increased immediately after light injury. The Rac1/Cdc42 effector PAK3 was highly expressed in photoreceptors, and its mRNA levels were increased after the onset of light-induced degeneration. A similar increase in PAK4 mRNA was also observed. In contrast, neither the distribution patterns of PAK 1 and PAK2 nor the mRNA levels of PAK 5 were altered during degeneration. 

Materials and Methods

All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Biomedical Sciences Institute/University of São Paulo Ethical Committee for Animal Research. 

Experimental Model and Tissue Preparation

Adult BALB/c mice (n = 90) were kept in a 12-hour light/12-hour dark cycle (light intensity, 80–100 lux) with food and water ad libitum and were dark adapted for 18 hours before exposure to constant light in a reflective cage. Immediately before stimulation, their pupils were dilated with 5% cyclopentolate (Allergan, São Paulo, Brazil); mice were then exposed to 2 hours of cool white fluorescent light at a luminescence level of 5000 lux. ³⁹ They were killed immediately after light exposure (L2) and after 6, 24, and 48 hours of darkness (D) that followed the 2-hour light exposure (L2D6, L2D24, and L2D48, respectively). 

Mice were deeply anesthetized with ketamine (12 mg/100 g body weight, intramuscularly; Parke-Davis, Ann Arbor, MI) and xylazine (0.8 mg/100 g intramuscularly, West Haven, CT) and underwent transcardial perfusion with saline followed by 2% paraformaldehyde in phosphate buffer (PB) 0.1 M (pH 7.4). Their eyes were dissected and cryoprotected in 30% sucrose in PB for at least 24 hours at 4°C. After they were embedded in OCT compound, retinas were sectioned perpendicularly to the vitreal surface on a cryostat (12-μm sections). In some experiments, eyes from postnatal day 10 animals (n = 3) were also used, as were mutant rd/rd mice of the CH3 strain (n = 3). 

TUNEL Technique for Apoptosis Detection

Apoptosis was detected by a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay, using a kit (Roche Molecular Biochemicals, Mannheim, Germany). Sections were coverslipped using VectaShield (Vector Laboratories, Burlingame, CA) and were observed under a confocal microscope (PCM 2000; Nikon, Tokyo, Japan). 

Immunohistochemistry

Retinal sections were incubated for 1 hour in a blocking solution containing 3% normal goat serum, 1% bovine serum albumin, and 0.3% Triton X-100 in PB. Primary rabbit antibodies against Rac1 (4 μg/mL; #sc-217) from Santa Cruz Biotechnology (Santa Cruz, CA), PAKs 1, 2 (1:100; #2602 and 2608, respectively) from Cell Signaling Technology (Beverly, MA) and PAK 3 (10 μg/mL; #AB3822, Chemicon, Temecula, CA) were diluted in PB containing 3% goat normal serum and 0.3% Triton X-100, and sections were incubated overnight at room temperature. Double-labeling studies were also performed using anti-Rac1 and goat antivimentin as a marker for Müller cells (1:250; AB1620; Chemicon, Temecula, CA). After several washes in PB, sections were incubated for 2 hours with antibodies against rabbit IgG tagged to Alexa TM 488 (1:1000; Molecular Probes, Eugene, OR) and against goat IgG tagged to TRITC (1:100; Jackson Laboratories, West Grove, PA) diluted in PB containing 0.3% Triton X-100. In some cases, immunohistochemistry for Rac1 was followed by TUNEL assay or peanut agglutinin (PNA) histochemistry. In the latter case, retinal sections were incubated for 30 minutes at room temperature with fluorescein-conjugated peanut agglutinin (20 μg/mL in PB; Vector Laboratories) to label cone-associated matrix. After washing, the tissue was mounted using VectaShield and analyzed in a confocal microscope. Figures were mounted with Adobe Photoshop 5.0 (Deneba Software, Miami, FL). Manipulation of the images was restricted to threshold and brightness adjustments to the whole image. Controls for the experiments consisted of the omission of primary antibodies; no staining was observed in these cases. 

Rac1 Activation Assay

Rac1 activation assay kit was used in these experiments (Cytoskeleton, Denver, CO). Briefly, retinas were dissected and homogenized in ice-cold lysis buffer. Cell lysates were cleared by centrifugation (5 minutes at 8000 rpm at 4°C), and the supernatant protein concentration was determined by the Bradford method. After incubation with PAK-PBD beads (containing the PAK domain that binds active Rac1 and Cdc42) for 1 hour at 4°C on a rotator, the beads were pelleted by centrifugation at 5000g for 3 minutes at 4°C. The pellet was resuspended in Laemmli buffer and submitted to electrophoresis (SDS-PAGE) in 15% gels. After transfer of the proteins to nitrocellulose filters, active Rac1 was detected using a polyclonal antibody (Santa Cruz Biotechnology). Detection of labeled proteins was achieved by using the enhanced chemiluminescence system (ECL; Amersham Biosciences, Buckinghamshire, England). Results are representative of two independent experiments. 

RNA Isolation, cDNA Synthesis, and Real-Time PCR

Total RNA was isolated from retinas homogenized in Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. Residual DNA was removed using DNase I (Amersham, Piscataway, NJ). For each 20-μL reverse transcription reaction, 4 μg total RNA was mixed with 1 μL (0.5 g) oligodT primer (Invitrogen) and was incubated for 10 minutes at 65°C. After cooling on ice, the solution was mixed with 4 μL of 5 × first-strand buffer, 2 μL of 0.1 M dithiothreitol (DTT), 1 μL deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP) (each 10 mM), and 1 μL (200 U) reverse transcriptase (SuperScript II; Invitrogen). Real-time PCR was carried out (5700 SDS Real-Time PCR machine; Applied Biosystems, Foster City, CA) with specific PAK3 [forward, 5′-AGG AGA TAA AAG TGC CCA TGG A-3′; reverse, 5′-CCA AAG GAG GTT CCG AAG CT-3′], PAK4 [forward, 5′-GGC GCC CTC ACG GAT ATT-3′; reverse, 5′-CAC GGC GGC GAT CTG T-3′], and PAK5 [forward, 5′-CAC TGT CAG GGT CCG ACA CTT AC-3′; reverse, 5′-GAG TAG CCT GCT TTG CTT TGA CT-3′] primers for mouse. Sequences were designed using Primer Express software (version 2.0; Applied Biosystems), synthesized and purified by high-performance liquid chromatography. All PCR assays were performed as follows: after initial activation at 50°C for 2 minutes and 95°C for 10 minutes, cycling conditions were 95°C for 10 seconds and 60°C for 1 minute Dissociation curves of PCR products were obtained by heating samples from 60°C to 95°C to test the specificity of the primers. Relative quantification of target gene expression was evaluated using the comparative CT method, as previously described in detail. ⁴⁰ The ΔCT value was determined by subtracting the target CT of each sample from its respective GAPDH CT value. Calculation of ΔΔCT involves using the control group mean ΔCT value as an arbitrary constant to subtract from all other ΔCT mean values. Fold-changes in gene expression of the target gene are equivalent to 2^−ΔΔCT. Values were entered into a one-way analysis of variance (ANOVA), followed by pairwise comparisons (Tukey’s HSD test), with significance level set at 5%. 

Results

Rac1 Expression after Photoreceptor Cell Death Induction

Rac1 immunoreactivity was mostly observed in the inner and outer segments of photoreceptors and in the plexiform layers (Fig. 1) . Rac1 expression was not restricted to a specific photoreceptor subtype because it was not always colocalized with PNA, a cone-specific marker (Fig. 1) . 

Although we did not specifically identify the origin of the Rac1 expression observed in the outer and inner plexiform layers, we believe expression might have been caused by the joint contribution of Rac1-positive photoreceptor, horizontal, and amacrine cells. In contrast, most of the Rac1-positive processes running toward the inner limiting membrane belonged to Müller glial cells, as confirmed by double labeling with an antibody against the intermediate filament protein vimentin (Fig. 1) . 

On degenerative light stimulus, a gradual reduction in outer nuclear layer thickness was observed because of the progressive death of photoreceptors, as seen by an increase in TUNEL-positive cells. The thickness of other retinal layers was not affected by light stimulus at the time points investigated. Light exposure also increased Rac1 expression in the outer nuclear layer (Fig. 2) . Diffuse and very weak Rac1 expression in the outer nuclear layers of control retinas progressively changed to strong and well-defined perikarya labeling in the L2D24 group. The rd1 mouse, another retinal degeneration model system in which photoreceptors die massively, also showed increased Rac1 expression in the outer nuclear layer during photoreceptor death at postnatal day 10 (Fig. 2) . 

Taken together, these data suggested that increased expression of Rac1 in the outer nuclear layer was related to light-induced photoreceptor death. To further investigate this hypothesis, we followed the progression of photoreceptor death and Rac1 expression by double-labeling experiments with TUNEL and Rac1. Figure 3shows that whereas all Rac1-positive cells were also TUNEL-positive since the early stages of degeneration, not all apoptotic cells showed expression of this GTPase, especially in the later stages of retinal degeneration. Furthermore, as the TUNEL signal increased (up to 48 hours after light stimulus), the size of Rac1-positive subpopulation remained constant from L2D24 to L2D48 (Fig. 3) . In later stages, a few Rac1-positive and TUNEL-negative cells were observed (Fig. 3) . 

Rac1 Activity Is Increased in the Retina after Light-Induced Degeneration

To verify whether Rac1 activity accompanied its expression increase, we used a pull-down assay with beads containing recombinant PAK domains. In agreement with its increased expression, the amount of GTP-bound Rac1 increased in the retina after light injury. However, when compared with the dark-adapted control group, Rac1 activity was already increased immediately after light stimulus (L2). This activity was maintained until L2D48 (Fig. 4) . Thus, light stimulus induced Rac1 activity before the increase in its expression. 

Light Stimulus Increases the Expression of PAK3 and PAK4

The distribution of PAKs 1 to 3 in control and light-injured retinas was investigated by immunofluorescence. PAK1 and PAK2 were observed in both plexiform layers, and, especially PAK1, in cell bodies of the inner nuclear layer and the ganglion cell layer of the control retina. In the whole retina, PAK1 expression was much higher than PAK2. Photoreceptor segments were labeled for PAK1 and PAK2, although PAK2 labeling was weak (Fig. 5) . After light stimulus, the distribution patterns of PAK1 and PAK2 did not change (Fig. 5) . 

PAK3 was present and extensively distributed in the outer and inner segments of photoreceptors and in the inner nuclear layer (possibly amacrine and bipolar cells), in both plexiform layers, and in the ganglion cell layer (Fig. 5) . Although the light stimulus did not change PAK3 distribution in the whole retina, labeling was slightly different, probably because of compaction of the protein in the outer segments caused by photoreceptor degeneration. 

We also analyzed PAK3, PAK4, and PAK5 gene expression by using real-time PCR, a quantitative reverse transcription–polymerase chain reaction that provides more precision and greater dynamic range than end point PCR. ⁴¹  

Real-time PCR quantitative analysis revealed that PAK gene expression profiles were distinct during retinal degeneration. As shown in Figure 6 , PAK3 mRNA levels in the L2D6 were similar to those of control but increased almost 20-fold (P < 0.001) in the L2D24 group. PAK4 mRNA in the L2D6 group was significantly more abundant when compared with control (2.9-fold; P < 0.01), maintaining a steady increase that was verified later in L2D24 (approximately 15.6-fold; P < 0.001). PAK5 gene expression, on the other hand, was not affected by light-degenerating stimulus. 

Discussion

Rho GTPases play a central role in cellular processes that involve the reorganization of the actin-based cytoskeleton, neuronal differentiation, cell cycle entry, and cell survival. ^{12 42} In the present study, Rac1 was observed in the inner and outer segments of photoreceptors. In these cells, the role of Rho GTPases in the maintenance of cell morphology ⁴³ and in rhodopsin signaling ^{38 44} has been previously investigated. For instance, in Drosophila rhodopsin–null mutants, the transgenic expression of constitutively active Rac1 rescued photoreceptor morphogenesis, whereas the expression of dominant-negative Rac1 resulted in retinal degeneration. ³¹ Recently, Deretic and coworkers ⁴⁵ suggested that this relationship is also maintained in vertebrate photoreceptors, showing that the trafficking of photopigments toward the rod outer segment is mediated by rhodopsin-bearing transport carriers and regulated by protein–lipid interactions that involve phosphatidylinositol-4,5-bisphosphate, moesin, actin, Rac1, and Rab8. 

An increase in Rac1 labeling in the outer nuclear layer in early light-damaged photoreceptors (L2D6 group) was observed, preceded by an increase in Rac1 activity in the whole retina. Rac1-expressing photoreceptors were also TUNEL-positive, suggesting a relationship between Rac1-increased expression and apoptosis. Data obtained from rd mice support such a hypothesis. Rd mice carry an autosomal-recessive mutation that leads to nearly complete loss of rod photoreceptors by approximately 21 days after birth. ⁵ Rac1 expression was also upregulated in these mice after the onset of photoreceptor degeneration at approximately postnatal day 10, further suggesting that Rac1 might participate in a common apoptotic pathway in those cells. 

In the L2D6 group, almost all TUNEL-positive cells in the outer nuclear layer were also labeled for Rac1; at L2D24, most cells were double labeled, but a few cells were only TUNEL-positive or Rac1-positive; at L2D48, the number of Rac1-positive cells remained the same, but TUNEL-positive cells increased. One possible explanation for this result is that TUNEL labeling was cumulative during L2D48, whereas Rac1 increased expression might have been transitory, preceding DNA-strand breaks labeled by the TUNEL technique. Additional experiments are necessary to check whether the former Rac1-positive cells died or whether Rac1 levels in those cells were reduced after a certain stage in cell death. 

The increase in Rac1 activity was already significant immediately after the luminous stimulus in the L2 group, thus preceding the increase in Rac1 expression, and remained high throughout the experiment (L2D48). Although Rac1 activity was measured in the whole retina, immunohistochemical analysis showed that increased Rac1 expression was restricted to photoreceptors only. Taken together, these results suggest that Rac1 activation is an early event in photoreceptor degeneration. 

Apoptosis mediated by Rac1 might have resulted from a signaling mechanism involving biochemical and transcriptional events. In different cell types, the production of reactive oxygen species (ROS) by the Rac1-NADPH oxidase system promotes cell death, ^{33 34 35 46} including drug-induced cell death. ^{47 48} The increased expression and activity of Rac1 in photoreceptors after light stimulus, in parallel with the massive ROS production observed in this degenerative model, ³⁹ suggest a correlation between these two events. Rac1 can also regulate gene expression often through the activation of kinase cascades leading to enhanced activity of stress-activated protein kinases, such as JNK, as shown, for example, in rat ventricular myocytes ⁴⁹ and in neurons. ⁵⁰  

Conversely, Rac1 activation might also have survival effects through the activation of phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB or Akt). ^{32 51 52 53 54 55 56} Kwon et al. ⁵⁷ provided evidence that Rac1 phosphorylation (and consequent inactivation) performed by Akt and stimulated by PI3-K is related to cell survival. In a model of oxidative stress and apoptosis induced by hypoxia/reoxygenation, the constitutive activation of Akt or PI3-K was sufficient to phosphorylate Rac1, inhibit Rac1 activation, and suppress Rac1-regulated ROS production and apoptosis. ³²  

In the present study, we also describe for the first time the distribution of PAKs 1, 2, and 3 in the mouse retina and the expression of PAKs 3, 4, and 5 mRNA before and after degenerative light stimulus. The potential regulation of stress responses and apoptosis by PAKs suggests that they might be therapeutically useful targets in a number of disease states. ⁵⁸  

Although group 1 PAKs (1, 2, and 3) were similar in structure, their distribution in the retina was very different. This was especially true for PAK3, which was highly expressed in the photoreceptor segments. After the onset of retinal degeneration, PAK3 mRNA increased drastically in the L2D24 group. A similar increase was observed for PAK4 mRNA, a protein with antiapoptotic functions in other cell types that increase the phosphorylation of the proapoptotic protein Bad and inhibit caspase activation. ^{59 60} A similar role was also attributed to PAK5. ⁶¹ However, we did not observe an increase in PAK5 expression after light stimulus. The increase in PAK4 mRNA levels was observed already in the L2D6 group and might have been related to the increased Rac1 activity. The fact that we did not observe an increase in PAK3 protein using immunohistochemistry might have been related to its initial intense expression in photoreceptors, and an additional increase might have surpassed the limits of the technique’s resolution. 

McPhie et al. ⁶² showed that neuronal apoptosis caused by Alzheimer disease mutants of the amyloid precursor protein (APP) are mediated by PAK3, which interacts with APP. A dominant-negative kinase mutant of PAK3 inhibited apoptosis, though it did not inhibit chemically induced apoptosis. Thus, it is possible that in our model, PAK3 acted downstream of Rac1 to induce photoreceptor death. 

Although apoptotic and anti-apoptotic roles have been attributed to PAK1 and PAK2, ^{63 64 65 66} their unchanged expression in the retina after light stimulus suggests that they do not participate in light-induced photoreceptor death. 

In conclusion, our study suggests that Rac1 might play an important role in the apoptosis of damaged photoreceptors, and potential downstream Rac1 effectors in this pathway, such as PAK3, are under investigation. During retinal remodeling, an increased expression of PAK4, potentially related to a defensive response, was also triggered in the retina. 

 Supported by Fundação de Amparo à Pesquisa no Estado de São Paulo (FAPESP #01/09047–2), and Conselho Nacional de Pesquisa (CNPq). MAB and AHK were recipients of fellowships from FAPESP.

 Submitted for publication April 9, 2005; revised August 7, August 13, and October 31, 2005; accepted December 28, 2005.

 Disclosure: M.A. Belmonte, None; M.F. Santos, None; A.H. Kihara, None; C.Y.I. Yan, None; D.E. Hamassaki, 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.

 

Rac1 expression in the mouse retina. Confocal micrographs of transverse retinal sections shows Rac1 (A, green), the intermediate filament vimentin (B, red), and their colocalization in Müller glial cell processes (C, arrows). Arrowheads indicate Rac1-labeled photoreceptor segments. Double-labeling with PNA (green) suggests that Rac1 (red) is not restricted to cones (E, arrowheads). (D, F) Control experiments with omission of the primary antibodies and PNA (F). os, photoreceptor outer segments; is, photoreceptor inner segments; onl, outer nuclear layer; opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer.

Figure 2.

 

- Rac1 expression in the outer retina of mice after light-induced retinal degeneration. Weak and diffuse Rac1 immunoreactivity can be seen in the outer nuclear layer of dark-adapted control mice (A) and mice killed immediately after light exposure (L2, B). Increased Rac1 expression can be observed 6 hours after light exposure (L2D6, C), and dramatic changes can be seen after 24 hours (L2D24, D). At that time, when photoreceptor segments were disrupted, many cell bodies expressed Rac1 (arrows). (D) Asterisks indicate retinal epithelium. Increased Rac1 expression was also detected in photoreceptor degeneration that occurred during development of the rd1 mouse (F) compared with the wt mouse (E) of the same age (postnatal day 10). is, photoreceptor inner segments; onl, outer nuclear layer; opl, outer plexiform layer. Scale bar, 30 μm.

Figure 3.

 

Rac1 expression by apoptotic cells. TUNEL-positive photoreceptors (green) were visible in the outer nuclear layer 6 hours after light exposure (L2D6, A) and increased after 24 hours (L2D24, D) and 48 hours (L2D48, G). Rac1-positive cells (red, B, E, and H) followed this pattern; almost all of them colocalized with TUNEL (arrows in C, F, I). Some TUNEL-positive cells that were not Rac1-positive, and the reverse, were also observed (arrowheads in F and I).

Figure 4.

 

Retinal degeneration stimulates Rac1 activity. Whole-retina extracts were precipitated with the p21-binding domain of PAK and blotted with anti-Rac1 antibody. C, dark-adapted control; L2, mice killed immediately after light exposure; L2D48, mice kept for 48 hours in darkness after 2-hour light exposure; GTPγS and GDP, positive and negative controls for the reaction, respectively.

Figure 5.

 

Immunohistochemistry for p21-activated kinases (PAKs) 1 to 3 in retinal sections of dark-adapted control mice (left) and mice killed 24 hours after the 2-hour light exposure (right). Conspicuous PAK3 immunoreactivity can be seen in photoreceptor segments compared with PAK1 and PAK2 immunoreactivities. Arrows show possible bipolar cells. os, photoreceptor outer segments; is, photoreceptor inner segments; onl, outer nuclear layer; opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer.

Figure 6.

 

p21-Activated kinases (PAKs) 3, 4, and 5 mRNA after light-induced retinal degeneration, using real-time PCR. PAK3 and PAK4, but not PAK5, mRNA increased dramatically in L2D24. C, dark-adapted control mice; L2D6, 6 hours in darkness after 2-hour light exposure; L2D24, 24 hours in darkness after 2-h light exposure. Bars represent SEM. *P < 0.1 versus control; **P < 0.01 versus control.

The authors thank Marley Januário da Silva and Leandro Mantovani de Castro for their excellent technical assistance.