October 2016
Volume 57, Issue 13
Open Access
Retinal Cell Biology  |   October 2016
Down-Regulation of RNA 3′-Terminal Phosphate Cyclase Attenuates Toll-Like Receptor 3-Mediated Axonal Loss in the Retina and Optic Nerve
Author Affiliations & Notes
  • Shravan K. Chintala
    Laboratory of Ophthalmic Neurobiology, Eye Research Institute of Oakland University, Rochester, Michigan, United States
  • Naveena Daram
    Laboratory of Ophthalmic Neurobiology, Eye Research Institute of Oakland University, Rochester, Michigan, United States
  • Correspondence: Shravan K. Chintala, Eye Research Institute of Oakland University, 118 Library Drive, 409 DHE, Rochester, MI 48309, USA; Chintala@oakland.edu
  • Footnotes
     Current affiliation: *Institute for Genetic Medicine, Keck School of Medicine, 2250 Alcazar Street, CSC-240, Los Angeles, CA 90033, USA; Chintals@usc.edu.
Investigative Ophthalmology & Visual Science October 2016, Vol.57, 5338-5347. doi:10.1167/iovs.16-19799
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      Shravan K. Chintala, Naveena Daram; Down-Regulation of RNA 3′-Terminal Phosphate Cyclase Attenuates Toll-Like Receptor 3-Mediated Axonal Loss in the Retina and Optic Nerve. Invest. Ophthalmol. Vis. Sci. 2016;57(13):5338-5347. doi: 10.1167/iovs.16-19799.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To investigate the role of RNA 3′-terminal phosphate cyclase (Rtca) in Toll-like receptor 3 (TLR3)-mediated loss of retinal ganglion cells (RGCs) and their axons.

Methods: Polyinosinic-polycytidylic acid (Poly[I:C]) or PBS was injected into the vitreous humor of C57BL/6J and Tlr3 knockout mice. C57BL/6J mouse eyes were treated with Rtca silencing RNA or control RNA, with or without PBS or Poly(I:C). At 24, 48, and 72 hours after treatments, RGC loss was determined with the brain-specific homeobox/POU domain protein 3a antibody, and axonal loss was assessed by using the neuronal class III beta-tubulin (Tuj1) antibody. Axonal loss in the optic nerves was determined by anterograde-labeling of Cholera Toxin B. Western blot assays were performed to determine TLR3, Rtca, c-jun N-terminal kinase 3 (JNK3), and phospho-JNK3 (pJNK3) levels, and immunohistochemistry assays were performed to determine the cells that synthesize Rtca.

Results: Poly(I:C) significantly up-regulated the protein levels of TLR3, Rtca, JNK3, and pJNK3 in the retina. Rtca levels were increased in RGCs, and an increase in Rtca levels promoted significant loss of RGCs and their axons. In Tlr3 knockout mouse retinas, Poly(I:C) failed to elevate Rtca, JNK3, and pJNK3 protein levels and did not promote significant axonal loss. Also, Rtca silencing RNA down-regulated Rtca, JNK3, and pJNK3 in C57BL/6J mouse retinas, and down-regulation of Rtca attenuated Poly(I:C)-mediated loss of RGCs and their axons.

Conclusions: The results presented in this study show that the activation of TLR3 promotes the loss of RGCs and their axons by elevating Rtca levels in the retina. Also, the results presented in this study show that Rtca regulates JNK3 expression in the retina.

Degeneration of RGCs and their axons leads to vision loss in a number of retinal degenerative diseases, including primary open-angle glaucoma (POAG), which affects 60 million people worldwide.1,2 However, the molecular mechanisms that promote the degeneration of RGCs and their axons under glaucomatous conditions remain unclear. A few studies have reported that Toll-like receptor 3 (TLR3), which was traditionally implicated in the activation of mammalian innate immune response,35 also plays a major role in oxidative stress-mediated neuronal degeneration.48 Until recently, it was unclear whether TLR3 played a role in the degeneration of RGCs in blinding diseases such as glaucoma, in which oxidative stress plays a major role. A previous study reported that the protein levels of Toll-like receptor 2, TLR3, and Toll-like receptor 4, and in particular TLR3 levels were elevated in the retinas obtained from human POAG donor eyes and in astrocytes isolated from glaucomatous retinas.9 However, it was unclear how TLR3 protein synthesized by astrocytes promoted the degeneration of RGCs and their axons in POAG. 
To determine whether the activation of TLR3 promotes the degeneration of RGCs, we injected polyinosinic-polycytidylic acid (Poly[I:C]), a double-stranded RNA mimic that specifically activates TLR3, into the vitreous humor of mouse eyes and found that the activation of TLR3 promoted RGC degeneration.10 By using a TLR3-specific inhibitor, we also found that the inhibition of TLR3 activation significantly attenuated the degeneration of RGCs.10 However, several questions remained unanswered, including whether the activation of TLR3 also promotes axonal loss in the retina and the optic nerve. Interestingly, a recent and only study to date reported that the elevated levels of Rtca promoted axonal loss in a mouse model of optic nerve crush.11 Rtca, identified first in extracts of HeLa cells and Xenopus nuclei, is an enzyme that catalyzes the conversion of a 3′-phosphate group to the 2′,3′-cyclic phosphodiester at the 3′ end of RNA.12,13 Although Rtca, conserved in bacteria, archaea, and vertebrates, has been suggested to be involved in cellular RNA processing,14 the biological functions of this cyclase remained unclear until the first study by Song et al.,11 which reported the role of Rtca in axonal degeneration. Therefore, by employing C57BL/6J and Tlr3 knockout mice, experiments in this study were performed to investigate whether (1) activation of TLR3,10 rather than physical injury to the optic nerve (crush), promotes axonal loss; (2) activation of TLR3 promotes axonal loss by elevating the protein levels of Rtca in the retina; and (3) deletion of Tlr3 or down-regulation of the Rtca protein attenuates axonal loss in the retina. 
Materials and Methods
Materials
Poly(I:C) (CAS number 31852-29-2) was obtained from InvivoGen (San Diego, CA, USA). Antibodies against TLR3 (sc-12509) and brain-specific homeobox/POU domain protein 3a (Brn3a) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rtca antibody (HPA-027982) was procured from Atlas Biologicals (Stockholm, Sweden). The c-jun N-terminal kinase 3 (JNK3; 2305) and phospho-JNK3 (pJNK3) (9251) antibodies were obtained from Novus Biologicals (Littleton, CO, USA). The neuronal class III beta-tubulin (Tuj1) antibody (801201) was obtained from Covance (Princeton, NJ, USA). The actin antibody (04-1116) was obtained from EMD Millipore (Billerica, MA, USA). For immunohistochemical analysis, appropriate secondary antibodies conjugated to AlexaFlour488 (green) and AlexaFlour568 (red) were obtained from Invitrogen (Carlsbad, CA, USA). For Western blot analysis, secondary antibodies conjugated to IRDye 700 or 800 were obtained from LI-COR (Lincoln, NE, USA). AlexaFlour 555-conjugated Cholera Toxin B (CTB) was obtained from Molecular Probes (Eugene, OR, USA). Rtca silencing RNA (siRNA) and control RNA were obtained from Invitrogen (Carlsbad, CA, USA). 
Preparation of Transit-TKO-siRNA Complexes
Five nanomoles of negative control RNA or predesigned siRNA for Rtca gene were combined with 5 μL of Transit-TKO (Mirus Biologicals, Madison, WI, USA) and 50 μL of RNAse-free water, and the solution was incubated for 20 minutes at room temperature. The solution was evaporated under vacuum, and the precipitate was dissolved in 20 μL of RNAse-free water. Two microliters of control RNA or Rtca siRNA were injected into the vitreous humor. 
Intravitreal Injections
All in vivo experiments on C57BL/6J and Tlr3 knockout mice (Jackson Laboratory, Bar Harbor, ME, USA) were performed under general anesthesia according to Oakland University's Institutional Animal Care and Usage Committee guidelines and the Association for Research in Vision & Ophthalmology statement for the use of Animals in Ophthalmology and Vision Research. Adult C57BL/6J and Tlr3 knockout mice (n = 6, three independent experiments) were anesthetized by an intraperitoneal injection of Ketamine (Zeotis, Inc., Kalamazoo, MI, USA; 50 mg/kg body weight) and Xylazine (Lloyd Laboratories, Shenandoah, IA, USA; 8 mg/kg body weight). After applying a drop of topical anesthetic agent, proparacaine, Poly(I:C) (2 μg/2 μL volume) or PBS (2 μL) was injected into the vitreous humors of the right eyes of each mouse by using a NanoFil syringe equipped with a 36-gauge beveled needle (World Precision Instruments, Sarasota, FL, USA).10 In separate experiments, C57BL/6J mouse eyes were also treated with control RNA or Rtca siRNA, with or without PBS or Poly(I:C). For the determination of axonal loss in the optic nerves, the mouse eyes were treated with an intravitreal injection of AlexaFlour555-conjugated CTB 24 hours before euthanasia. 
CTB Labeling of Optic Nerve Axons
The loss of axons in the optic nerve was determined by anterograde labeling of AlexaFlour 555-conjugated CTB. At each time point, 1 μL of CTB (10 μg/μL) was injected into the vitreous humor of C57BL/6J and Tlr3 knockout mice before euthanasia (n = 6, three independent experiments). Optic nerves were then isolated from enucleated eyes and embedded in an OCT compound. The 10-μm thick radial sections were prepared by using a cryostat, and CTB labeling in the optic nerves was assessed by observing the sections under a Zeiss Imager Z.2 microscope (Zeiss, Thornwood, NY, USA). 
Protein Extraction
At 24, 48, and 72 hours after intravitreal injecting of control RNA or Rtca siRNA, with or without PBS or Poly(I:C), the retinal proteins were extracted as previously described (n = 6, three independent experiments).10 Protein concentration in retinal extracts was determined by using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). 
Western Blot Analysis
Aliquots containing an equal amount of retinal proteins (50 μg) extracted from eyes treated with control RNA or Rtca siRNA, with or without PBS or Poly(I:C), were separated on 10% SDS-PAGE (n = 6, three independent experiments). Proteins were transferred onto Immobilon-FL membranes, and nonspecific binding was blocked with an Odyssey blocking buffer (LI-COR, Lincoln, NE, USA) containing 0.2% Tween-20. After incubating with primary antibodies against Rtca, TLR3, JNK3, pJNK3, and actin, the membranes were probed with the appropriate secondary antibodies conjugated to IRDye 800 or 700 for 1 hour at room temperature. The membranes were scanned by using the Odyssey two-channel IR-detection scanner (LI-COR) to determine the relative levels of Rtca, TLR3, JNK3, and pJNK3. 
Immunohistochemistry
After intravitreal injection of control RNA or Rtca siRNA, with or without PBS or Poly(I:C) (n = 6 eyes, three independent experiments), the retinas were isolated and processed as whole retinas or embedded in an OCT compound for the preparation of cross-sections. The 10-μm thick cross-sections were prepared by using a cryostat according to previously published methods.10 Retinal cross-sections and whole retinas were immunostained with antibodies for Rtca or Tuj1, and digitized images were obtained by using a Zeiss Imager Z.2 microscope (Zeiss). 
Statistical Analysis
Statistical significance was determined by analysis of variance followed by a post hoc Tukey's test (GB-Stat Software, Dynamic Microsystems, Silver Spring, MD, USA). 
Results
TLR3 Activation Promotes the Loss of RGCs and Their Axons in the Retina
To determine whether the activation of TLR3 promotes the loss of RGC somas and their axons in the retina, Poly(I:C) or PBS was injected into the vitreous humor of C57BL/6J mouse eyes (n = 6; three independent experiments). At 24, 48, and 72 hours after the treatments, whole retinas were isolated and immunostained with antibodies against Brn3a (a marker for RGCs) and Tuj1, a marker for axons (Fig. 1A). The number of remaining RGCs (loss of Brn3a staining) and axons (loss of Tuj1 staining) in eight areas of equal size (Fig. 1B) located at an equal distance from the optic disc were quantified. The results presented in Figure 1A show that when compared with PBS-treated retinas, Poly(I:C) promoted progressive loss of Brn3a-positive RGCs and Tuj1-positive axons during the 72-hour period. RGC quantification data in Figure 1C show that when compared with 424 ± 25.05 Brn3a-positive cells in the retinas from PBS-treated eyes, Poly(I:C) promoted a significant loss of Brn3a-positive RGCs at 24 (246.66 ± 45.09), 48 (170 ± 17.32), and 72 hours (101.66 ± 7.63). #P < 0.015, when Brn3a-positive RGCs in Poly(I:C)-treated retinas were compared with PBS-treated retinas. Axonal tracing data in Figure 1D show that when compared with 50 ± 6.73 axons in the PBS-treated eyes, Poly(I:C) promoted a significant loss of axons at 24 (32.5 ± 3.78), 48 (17.75 ± 2.06), and 72 hours (9.75 ± 2.63). *P < 0.02, when Tuj1-positive axons in Poly(I:C)-treated retinas were compared with PBS-treated retinas. 
Figure 1
 
Loss of RGCs and their axons in CB57BL/6J mouse retinas. At 24, 48, and 72 hours after PBS or Poly(I:C)-treatment, RGC loss was determined by immunostaining of whole retinas with Brn3a antibody (A, leftmost panels) and axonal loss was determined with Tuj1 antibody (A, middle panels). After immunostaining, the number of remaining RGCs and axons in eight areas of equal size (B), located at an equal distance from the optic disc were quantified by using NIH ImageJ software. Bar graph C shows the quantification of remaining RGCs, and bar graph D shows the remaining axons at each time point. #P < 0.015 when Brn3a-positive RGCs in Poly(I:C)-treated retinas were compared with PBS-treated retinas. *P < 0.02 when Tuj1-positive axons in Poly(I:C)-treated retinas were compared with axons in PBS-treated retinas.
Figure 1
 
Loss of RGCs and their axons in CB57BL/6J mouse retinas. At 24, 48, and 72 hours after PBS or Poly(I:C)-treatment, RGC loss was determined by immunostaining of whole retinas with Brn3a antibody (A, leftmost panels) and axonal loss was determined with Tuj1 antibody (A, middle panels). After immunostaining, the number of remaining RGCs and axons in eight areas of equal size (B), located at an equal distance from the optic disc were quantified by using NIH ImageJ software. Bar graph C shows the quantification of remaining RGCs, and bar graph D shows the remaining axons at each time point. #P < 0.015 when Brn3a-positive RGCs in Poly(I:C)-treated retinas were compared with PBS-treated retinas. *P < 0.02 when Tuj1-positive axons in Poly(I:C)-treated retinas were compared with axons in PBS-treated retinas.
Activation of TLR3 Promotes Axonal Loss in the Optic Nerve
To determine whether the activation of TLR3 promotes axonal loss in the optic nerve, C57BL/6J mouse eyes were treated with intravitreal injections of Poly(I:C) or PBS. One day before euthanizing the mice, axons in the optic nerves were labeled anterogradely with AlexaFlour 555-conjugated CTB. At 24, 48, and 72 hours after the treatments, 10-μ thick radial sections of the optic nerves were prepared, and CTB labeling was determined by observing the sections under a fluorescence microscope. The data presented in Figures 2A and 2B show that when compared with CTB-labeling distance in the optic nerves from PBS-treated eyes (600 ± 20 μm), CTB-labeling distance was reduced significantly in the optic nerves isolated from Poly(I:C)-treated mouse eyes at 24 (500 ± 25 μm), 48 (300 ± 40 μ), and 72 hours (125 ± 25 μm). When CTB-labeling distance in the optic nerves isolated from Poly(I:C)-treated eyes was compared with CTB-labeling distance in the optic nerves isolated from PBS-treated eyes, P < 0.01. 
Figure 2
 
Axonal loss in the optic nerves of C57BL/6J mice. One day before euthanizing the mice that were treated with PBS or Poly(I:C), axons in the optic nerves were anterogradely labeled with AlexaFlour 555–conjugated CTB. At 24, 48, and 72 hours after the treatments, radial sections of the optic nerves were prepared and imaged by using a fluorescence microscope (A). Data presented in B show a progressive decrease of CTB-labeling in the optic nerves isolated from Poly(I:C)-treated eyes when compared with the optic nerves isolated from PBS-treated eyes. *P < 0.01 when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with CTB-labeling in the optic nerves from PBS-treated eyes.
Figure 2
 
Axonal loss in the optic nerves of C57BL/6J mice. One day before euthanizing the mice that were treated with PBS or Poly(I:C), axons in the optic nerves were anterogradely labeled with AlexaFlour 555–conjugated CTB. At 24, 48, and 72 hours after the treatments, radial sections of the optic nerves were prepared and imaged by using a fluorescence microscope (A). Data presented in B show a progressive decrease of CTB-labeling in the optic nerves isolated from Poly(I:C)-treated eyes when compared with the optic nerves isolated from PBS-treated eyes. *P < 0.01 when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with CTB-labeling in the optic nerves from PBS-treated eyes.
TLR3 Activation Up-Regulates Rtca and TLR3 Protein Levels in the Retina
To determine whether Poly(I:C)-mediated loss of RGCs and their axons is a result of elevated levels of Rtca protein through the activation of TLR3, C57BL/6J mouse eyes were treated with intravitreal injections of PBS or Poly(I:C). Western blot analysis was performed on retinal proteins extracted at 24, 48, and 72 hours after the treatments by using antibodies for Rtca and TLR3. The results presented in Figures 3A and 3B show that when compared with low levels of TLR3 protein in retinal proteins extracted from PBS-treated eyes (12.25 ± 2.62 arbitrary units [AU]), TLR3 levels were significantly up-regulated in retinal proteins extracted from Poly(I:C)-treated eyes at 24 (32.0 ± 2.82 AU), 48 (56.75 ± 5.37 AU), and 72 hours (70.75 ± 2.98 AU). In addition, when compared with low levels of Rtca protein in retinal proteins extracted from PBS-treated eyes (14.75 ± 0.95 AU), Rtca protein levels were significantly elevated in retinal proteins extracted from Poly(I:C)-treated eyes at 24 (26.75 ± 2.06 AU), 48 (58.25 ± 2.36 AU), and 72 hours (48.0 ± 1.63 AU). *P < 0.03, when TLR3 and Rtca protein levels in Poly(I:C)-treated retinas were compared with TLR3 and Rtca protein levels in PBS-treated retinas. 
Figure 3
 
Up-regulation of Rtca protein in the retinas of C57BL/6J mice. (A) Retinal proteins extracted from PBS or Poly(I:C)-treated eyes (50 μg) were subjected to Western blot analysis by using antibodies for Rtca and TLR3. Actin antibody was used to determine equal loading of the proteins. (B) The bar graph shows the relative levels of Rtca and TLR3 in retinal protein extracts. Results presented in the figure show that Poly(I:C) up-regulated the protein levels of both Rtca and TLR3 in the retina. *P < 0.03 when TLR3 and Rtca protein levels in Poly(I:C)-treated retinas were compared with TLR3 and Rtca protein levels in PBS-treated retinas.
Figure 3
 
Up-regulation of Rtca protein in the retinas of C57BL/6J mice. (A) Retinal proteins extracted from PBS or Poly(I:C)-treated eyes (50 μg) were subjected to Western blot analysis by using antibodies for Rtca and TLR3. Actin antibody was used to determine equal loading of the proteins. (B) The bar graph shows the relative levels of Rtca and TLR3 in retinal protein extracts. Results presented in the figure show that Poly(I:C) up-regulated the protein levels of both Rtca and TLR3 in the retina. *P < 0.03 when TLR3 and Rtca protein levels in Poly(I:C)-treated retinas were compared with TLR3 and Rtca protein levels in PBS-treated retinas.
Activation of TLR3 Leads to Up-Regulation of Rtca in RGCs
To determine whether the activation of TLR3 up-regulates Rtca protein levels in RGCs, retinal cross-sections prepared at 24, 48, and 72 hours after treating C57BL/6J mice with Poly(I:C) or PBS were immunostained with an antibody for Rtca and double labeled with Tuj1 antibody, a marker for RGCs and their axons. The results presented in Figure 4 show that when compared with undetectable levels of Rtca protein in retinal cross-sections prepared from PBS-treated eyes, Rtca protein levels were increased in the retinal sections prepared from Poly(I:C)-treated eyes at 24, 48, and 72 hours, and increased levels of Rtca were colocalized in Tuj1-positive RGCs. 
Figure 4
 
Localization of Rtca protein in RGCs of C57BL/6J mice. At 24, 48, and 72 hours after PBS or Poly(I:C)-treatment, retinal cross-sections prepared from C57BL/6J mouse eyes were immunostained by using an antibody against Rtca and double labeled with Tuj1 antibody. Results presented in the figure show that Rtca protein, absent in cross-sections prepared from PBS-treated eyes, was elevated in retinal cross-sections prepared from Poly(I:C)-treated eyes and colocalized in Tuj1-positive RGCs (red arrows). The white bar indicates a size of 50 μm.
Figure 4
 
Localization of Rtca protein in RGCs of C57BL/6J mice. At 24, 48, and 72 hours after PBS or Poly(I:C)-treatment, retinal cross-sections prepared from C57BL/6J mouse eyes were immunostained by using an antibody against Rtca and double labeled with Tuj1 antibody. Results presented in the figure show that Rtca protein, absent in cross-sections prepared from PBS-treated eyes, was elevated in retinal cross-sections prepared from Poly(I:C)-treated eyes and colocalized in Tuj1-positive RGCs (red arrows). The white bar indicates a size of 50 μm.
Rtca Was Not Elevated in Poly(I:C)-Treated Tlr3 Knockout Mouse Retinas
Because TLR3-mediated up-regulation of Rtca correlated with axonal loss in C57BL/6J mouse retinas (Fig. 3), experiments were performed to determine the causal role of TLR3 in Rtca expression. C57BL/6J and Tlr3 knockout mouse eyes (n = 6, three independent experiments) were treated with PBS or Poly(I:C). At 48 hours after the treatment, Western blot analysis was performed on retinal proteins extracted from C57BL/6J and Tlr3 knockout mouse eyes to determine the relative levels of the Rtca protein (Fig. 5A). The results presented in Figure 5B show that when compared with low levels of the Rtca protein in retinal proteins extracted from PBS-treated C57BL/6J mouse eyes (4.25 ± 1.70 AU), Rtca protein levels were significantly elevated in the retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes (54.25 ± 3.31 AU). In contrast, despite Poly(I:C) treatment, Rtca protein levels were not elevated in the retinal proteins extracted from Tlr3 knockout mice (3.5 ± 0.57 AU) when compared with Rtca protein levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes. *P < 0.02, when Rtca levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes were compared with Rtca levels in retinal proteins extracted from PBS-treated C57BL/6J mouse eyes. **P < 0.03, when Rtca levels in retinal proteins extracted from Poly(I:C)-treated Tlr3 knockout mouse eyes were compared with Rtca levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes. 
Figure 5
 
Rtca, JNK3, and pJNK3 levels were not up-regulated in Tlr3 knockout mice. At 48 hours after treating C57BL/6J and Tlr3 knockout mouse eyes with PBS or Poly(I:C), Western blot analysis was performed on total proteins extracted from the retinas by using Rtca, JNK3, and pJNK3 antibodies (A). Actin bands show the loading of an equal amount of total proteins. The bar graph in B shows semiquantitative analysis of Rtca, JNK3, and pJNK3 levels from the Western blots. *P < 0.02 when Rtca, JNK3, and pJNK3 levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes were compared with Rtca, JNK3, and pJNK3 in retinal proteins extracted from PBS-treated C57BL/6J mouse eyes. **P < 0.03 when Rtca, JNK3, and pJNK3 levels in retinal protein extracted from Poly(I:C)-treated Tlr3 knockout mice were compared with Poly(I:C)-treated C57BL/6J mouse eyes.
Figure 5
 
Rtca, JNK3, and pJNK3 levels were not up-regulated in Tlr3 knockout mice. At 48 hours after treating C57BL/6J and Tlr3 knockout mouse eyes with PBS or Poly(I:C), Western blot analysis was performed on total proteins extracted from the retinas by using Rtca, JNK3, and pJNK3 antibodies (A). Actin bands show the loading of an equal amount of total proteins. The bar graph in B shows semiquantitative analysis of Rtca, JNK3, and pJNK3 levels from the Western blots. *P < 0.02 when Rtca, JNK3, and pJNK3 levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes were compared with Rtca, JNK3, and pJNK3 in retinal proteins extracted from PBS-treated C57BL/6J mouse eyes. **P < 0.03 when Rtca, JNK3, and pJNK3 levels in retinal protein extracted from Poly(I:C)-treated Tlr3 knockout mice were compared with Poly(I:C)-treated C57BL/6J mouse eyes.
In a previous study, we reported that a TLR3-specific inhibitor attenuated RGC loss by down-regulating the JNK3 protein.10 Therefore, to determine whether JNK3 protein levels were also reduced in Tlr3 knockout mice, retinal protein extracted from PBS or Poly(I:C)-treated C57BL/6J and Tlr3 knockout mice were subjected to Western blot analysis by using them against JNK3 and pJNK3. The results presented in Figures 5A and 5B show that when compared with low levels of JNK3 (25.0 ± 5.0 AU) and pJNK3 (3.33 ± 0.57 AU) in retinal proteins extracted from PBS-treated C57BL/6J mice, JNK3 and pJNK3 levels were significantly elevated in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mice (58.33 ± 7.73 and 48.33 ±7.63 AU, respectively). In contrast, Poly(I:C) failed to up-regulate JNK3 and pJNK3 levels in retinal proteins extracted from Tlr3 knockout mice (7.66 ± 2.51 and 4.66 ± 1.15 AU, respectively) when compared with JNK3 protein levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes. *P < 0.02, when JNK3 protein levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes were compared with Rtca levels in retinal proteins extracted from PBS-treated C57BL/6J mouse eyes. **P < 0.03, when JNK3 protein levels in retinal proteins extracted from Poly(I:C)-treated Tlr3 knockout mice were compared with Poly(I:C)-treated C57BL/6J mouse eyes. 
Down-Regulation of Rtca in Tlr3 Knockout Mice Attenuates Poly(I:C)-Mediated Loss of RGCs and Their Axons
To determine whether the down-regulation of Rtca in Tlr3 knockout mice observed previously (Fig. 5) also inhibited the loss of RGCs and their axons, Tlr3 knockout mouse eyes (n = 6, three independent experiments) were treated with PBS or Poly(I:C). At 48 hours after the treatment, the loss of RGCs in the retina was determined by immunostaining whole retinas with the Brn3a antibody, and the loss of axons in the retina was determined by immunostaining whole retinas with the Tuj1 antibody (Fig. 6A). In addition, axonal loss in the optic nerves was determined by anterograde labeling with CTB (Figs. 6D and 6E). The results presented in Figures 6A (top two panels) and 6B show a similar number of Brn3a-positive RGCs in the retinas isolated from PBS or Poly(I:C)-treated eyes (411.66 ± 10.40 vs. 402.66 ± 14.18, respectively), indicating that Poly(I:C) did not promote the loss of RGCs in the retinas of Tlr3 knockout mice. The results presented in Figures 6A (middle and bottom two panels) and 6C also show a similar number of axons in the retinas isolated from PBS or Poly(I:C)-treated retinas (50 ± 4.08 vs. 49.75 ± 7.32, respectively), indicating that Poly(I:C) did not promote axonal loss in the retinas of Tlr3 knockout mice. In addition, Poly(I:C) failed to promote axonal loss in the optic nerves of Tlr3 knockout mice (Figs. 6D and 6E) as observed by the similar distance of anterogradely labeled CTB in the optic nerves isolated from Poly(I:C)-treated Tlr3 knockout mouse eyes (600 ± 32.14) when compared with PBS-treated mouse eyes (600 ± 50). 
Figure 6
 
Loss of RGCs and their axons is attenuated in Tlr3 knockout mouse retinas. At 48 hours after treating Tlr3 knockout mouse eyes with PBS or Poly(I:C), axonal loss in the retina was determined by immunostaining of whole retinas with Tuj1 antibody. The results presented in A and B indicate that Poly(I:C) did not promote axonal loss in Tlr3 knockout mouse retinas when compared with the axons in PBS-treated Tlr3 knockout mouse eyes. NS, not significant when the number of axons in Poly(I:C)-treated retinas was compared with PBS-treated retinas. To determine axonal loss in the optic nerves, 24 hours before euthanizing PBS or Poly(I:C)-treated Tlr3 knockout mice, axons in the optic nerves were anterogradely labeled with CTB. At 24 hours after CTB labeling, mice were euthanized, radial sections of the optic nerves were prepared, and CTB-labeling in optic nerves was assessed by observing them under a fluorescence microscope (C). Results presented in C and D indicate that Poly(I:C) did not promote axonal loss in the optic nerves from Tlr3 knockout mice. NS, not significant when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS-treated eyes.
Figure 6
 
Loss of RGCs and their axons is attenuated in Tlr3 knockout mouse retinas. At 48 hours after treating Tlr3 knockout mouse eyes with PBS or Poly(I:C), axonal loss in the retina was determined by immunostaining of whole retinas with Tuj1 antibody. The results presented in A and B indicate that Poly(I:C) did not promote axonal loss in Tlr3 knockout mouse retinas when compared with the axons in PBS-treated Tlr3 knockout mouse eyes. NS, not significant when the number of axons in Poly(I:C)-treated retinas was compared with PBS-treated retinas. To determine axonal loss in the optic nerves, 24 hours before euthanizing PBS or Poly(I:C)-treated Tlr3 knockout mice, axons in the optic nerves were anterogradely labeled with CTB. At 24 hours after CTB labeling, mice were euthanized, radial sections of the optic nerves were prepared, and CTB-labeling in optic nerves was assessed by observing them under a fluorescence microscope (C). Results presented in C and D indicate that Poly(I:C) did not promote axonal loss in the optic nerves from Tlr3 knockout mice. NS, not significant when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS-treated eyes.
Rtca siRNA Decreases Poly(I:C)-Mediated Up-Regulation of Rtca, JNK3, and pJNK3 in the Retinas of C57BL/6J Mice
Although the results presented in Figure 5 indicate that the levels of Rtca protein were down-regulated in Tlr3 knockout mice despite Poly(I:C) treatment, these results simply suggest that TLR3 is responsible for the up-regulation of Rtca, but they do not indicate the causal role of Rtca in axonal loss. Therefore, to determine the causal role of Rtca in axonal loss, C57BL/6J mouse eyes were treated with intravitreal injections of control RNA or Rtca siRNA, with or without PBS or Poly(I:C). At 48 hours after the treatments, the retinal proteins were extracted and subjected to Western blot analysis by using the Rtca antibody. The results presented in Figure 7 show that when compared with low levels of Rtca in retinal proteins extracted from PBS-treated mice (5.00 ± 1 AU), the Rtca levels were elevated significantly in the Poly(I:C) treated eyes (56.66 ± 2.88 AU), consistent with the results presented in Figure 3. The Rtca levels remained unchanged in the retinal proteins extracted from PBS plus control RNA-treated (4.66 ± 0.5 AU) or PBS plus Rtca siRNA-treated eyes (5.66 ± 0.57 AU). In contrast, when compared with the elevated levels of Rtca protein in retinal proteins extracted from Poly(I:C)-treated eyes (56.66 ± 2.88 AU), the Rtca levels were down-regulated significantly in retinal proteins extracted from Poly(I:C) plus Rtca siRNA-treated eyes (5.33 ± 0.57 AU), but not in Poly(I:C) plus control RNA-treated mice (55 ± 5 AU). *P < 0.04, #P < 0.02, when Rtca levels in retinal proteins extracted from Poly(I:C)-treated eyes were compared with Rtca levels in retinal proteins extracted from PBS-treated eyes. **P < 0.03, when Rtca levels in retinal proteins extracted from Poly(I:C) plus Rtca siRNA-treated eyes were compared with Rtca levels in Poly(I:C) or Poly(I:C) plus control RNA-treated eyes. 
Figure 7
 
Down-regulation of Rtca, JNK3, and pJNK3 in the retinas from Rtca siRNA-treated C57BL/6J mice. Equal amounts of retinal proteins (50 μg) extracted from the eyes treated with control RNA or Rtca siRNA, with or without PBS or Poly(I:C) were subjected to Western blot analysis by using antibodies for Rtca, JNK3, and pJNK3 (A). Actin bands indicate loading controls for the total proteins. The bar graph (B) shows the relative levels of Rtca, JNK3, and pJNK3 in retinal protein extracts. *P < 0.03 and #P < 0.04 when Rtca, JNK3, and pJNK3 levels in the retinal proteins extracted from Poly(I:C)-treated eyes were compared with Rtca, JNK3, and pJNK3 levels in the retinal proteins extracted from PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.02 when Rtca, JNK3, and pJNK3 levels in retinal proteins extracted from Poly(I:C) plus Rtca siRNA-treated eyes were compared with Rtca, JNK3, and pJNK3 levels in Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Figure 7
 
Down-regulation of Rtca, JNK3, and pJNK3 in the retinas from Rtca siRNA-treated C57BL/6J mice. Equal amounts of retinal proteins (50 μg) extracted from the eyes treated with control RNA or Rtca siRNA, with or without PBS or Poly(I:C) were subjected to Western blot analysis by using antibodies for Rtca, JNK3, and pJNK3 (A). Actin bands indicate loading controls for the total proteins. The bar graph (B) shows the relative levels of Rtca, JNK3, and pJNK3 in retinal protein extracts. *P < 0.03 and #P < 0.04 when Rtca, JNK3, and pJNK3 levels in the retinal proteins extracted from Poly(I:C)-treated eyes were compared with Rtca, JNK3, and pJNK3 levels in the retinal proteins extracted from PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.02 when Rtca, JNK3, and pJNK3 levels in retinal proteins extracted from Poly(I:C) plus Rtca siRNA-treated eyes were compared with Rtca, JNK3, and pJNK3 levels in Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
The results presented in Figure 5 show that the levels of Rtca and JNK3 were down-regulated in Poly(I:C)-treated Tlr3 knockout mice, suggesting the possibility that TLR3 modulates the expression of both proteins. Therefore, to determine whether the down-regulation of Rtca by Rtca siRNA shown in Figure 5 also reduces JNK3 and pJNK3 levels, retinal proteins extracted from mice treated with control RNA or Rtca siRNA, with or without Poly(I:C), were subjected to Western blot analysis by using antibodies against JNK3 and pJNK3 (Fig. 7A). The results presented in Figure 7 indicate that when compared with low levels of JNK3 and pJNK3 in retinal proteins extracted from PBS-treated eyes (10.33 ± 4.50 51 and 4.66 ± 0.57 AU, respectively), JNK3 and pJNK3 levels were significantly elevated in the retinal proteins extracted from Poly(I:C)-treated eyes (31.66 ± 2.88 and 55 ± 5 AU, respectively), consistent with the results presented in Figure 5. The low levels of JNK3 and pJNK3 observed in the retinal proteins extracted from PBS-treated eyes (10.33 ± 4.50 and 4.66 ± 0.57 AU, respectively) remained similar in retinal proteins extracted from PBS plus control RNA-treated (9.66 ± 4.72 and 5.66 ± 1.52 AU, respectively) and PBS plus Rtca siRNA-treated eyes (9.33 ± 4.16 and 5.33 ± 0.57 AU, respectively). In contrast, when compared with the elevated levels of JNK3 and pJNK3 in the retinal proteins extracted from Poly(I:C)-treated eyes (31.66 ± 2.88 and 55 ± 5 AU, respectively), JNK3 and pJNK3 levels were significantly down-regulated in retinal proteins extracted from Poly(I:C) plus Rtca siRNA-treated eyes (8.66 ± 1.15 and 10.33 ± 1.5 AU, respectively), but not in Poly(I:C) plus control RNA-treated eyes (30.66 ± 1.15 and 53.66 ± 3.21 AU, respectively). These results indicate that Rtca regulates TLR3-mediated expression of JNK3 and pJNK3. *P < 0.03 and **P < 0.04, when JNK3 and pJNK3 levels in retinal proteins extracted from Poly(I:C)-treated eyes were compared with JNK3 and pJNK3 levels in retinal proteins extracted from PBS-treated eyes. **P < 0.02, when JNK3 levels in retinal proteins extracted from Poly(I:C) plus Rtca siRNA-treated eyes were compared with JNK3 levels in Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.  
Rtca siRNA Attenuates Poly(I:C)-Mediated Loss of RGCs and Their Axons in C57BL/6J Mouse Retinas
Because Rtca protein levels were down-regulated in Rtca siRNA plus Poly(I:C)-treated mouse retinas, experiments were performed further to determine whether the down-regulation of Rtca attenuates Poly(I:C)-mediated loss of RGCs and their axons in the retina. C57BL/6J mice were treated with intravitreal injections of control RNA or Rtca siRNA, with or without PBS or Poly(I:C). At 48 hours after the treatments, whole retinas were isolated and immunostained with the Brn3a antibody (Fig. 8A, leftmost panels). The remaining number of Brn3a-positive RGCs was quantified by using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health [NIH], Bethesda, MD, USA; Fig. 8B). The results presented in Figures 8A and 8B show that when compared with 416 ± 15 Brn3a-positive RGCs in the retinas isolated from PBS-treated eyes, the RGC number was reduced to 160 ± 10 in the retinas isolated from Poly(I:C)-treated eyes. #P < 0.04, when the number of Brn3a-positve RGCs in Poly(I:C)-treated retinas was compared with PBS-treated retinas. The number of Brn3a-positve RGCs remained similar in the retinas isolated from Poly(I:C) plus control RNA-treated eyes (158 ± 7.3) when compared with Poly(I:C)-treated eyes. In contrast, when compared with the number of Brn3a-positve RGCs in Poly(I:C) or Poly(I:C) plus control RNA-treated eyes, the number of Brn3a-positve RGCs was increased in the retinas isolated from Poly(I:C) plus Rtca siRNA-treated eyes (356 ± 30). ##P < 0.03, when the number of Brn3a-positive RGCs in Poly(I:C) plus Rtca siRNA-treated eyes was compared with Poly(I:C)-treated eyes.  
Figure 8
 
Inhibition of RGC and axonal loss in the retinas of Rtca siRNA-treated C57BL/6J mice. At 48 hours after treating C57BL/6J mouse eyes with control RNA or Rtca siRNA, with or without PBS or Poly(I:C), RGC loss was determined by immunostaining whole retinas with Brn3a antibody (A, left panels) and axonal loss was determined with Tuj1 antibody (A, middle panels). After immunostaining, the number of remaining RGCs (B) and their axons (C) in the retinas was quantified by using NIH ImageJ software. #P < 0.04, when the number of Brn3a-positive RGCs in Poly(I:C)-treated retinas was compared with PBS-treated retinas. ##P < 0.03 when the number of Brn3a-positive RGCs in Poly(I:C) plus Rtca siRNA-treated retinas was compared with Poly(I:C)-treated retinas. *P < 0.04 when Tuj1-positive axons in Poly(I:C)-treated eyes were compared with PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.03 when Tuj1-positive axons in Poly(I:C) plus Rtca siRNA-treated eyes were compared with Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Figure 8
 
Inhibition of RGC and axonal loss in the retinas of Rtca siRNA-treated C57BL/6J mice. At 48 hours after treating C57BL/6J mouse eyes with control RNA or Rtca siRNA, with or without PBS or Poly(I:C), RGC loss was determined by immunostaining whole retinas with Brn3a antibody (A, left panels) and axonal loss was determined with Tuj1 antibody (A, middle panels). After immunostaining, the number of remaining RGCs (B) and their axons (C) in the retinas was quantified by using NIH ImageJ software. #P < 0.04, when the number of Brn3a-positive RGCs in Poly(I:C)-treated retinas was compared with PBS-treated retinas. ##P < 0.03 when the number of Brn3a-positive RGCs in Poly(I:C) plus Rtca siRNA-treated retinas was compared with Poly(I:C)-treated retinas. *P < 0.04 when Tuj1-positive axons in Poly(I:C)-treated eyes were compared with PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.03 when Tuj1-positive axons in Poly(I:C) plus Rtca siRNA-treated eyes were compared with Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
To determine whether down-regulation of Rtca also attenuates axonal loss, whole retinas were immunostained with the Tuj1 antibody (Fig. 8A, middle panels). The remaining number of axons were traced manually and quantified by using NIH ImageJ software (Fig. 8A, right panels). The results presented in Figures 8A and 8C show that when compared with 47.5 ± 2.88 axons in the retinas isolated from PBS-treated eyes, the number of axons was reduced to 21 ± 1.82 in the retinas isolated from Poly(I:C)-treated eyes. The number of axons remained similar in the retinas isolated from Poly(I:C) plus control RNA-treated eyes (19 ± 1.41). In contrast, when compared with the number of axons in the retinas isolated from Poly(I:C) (21 ± 1.82) or Poly(I:C) plus control RNA-treated eyes (19 ± 1.41), an increased number of axons were found in the retinas isolated from Poly(I:C) plus Rtca siRNA-treated eyes (40.75 ± 4.57). These results indicated that Rtca played a causal role in the TLR3-mediated loss of RGCs and their axons in the retina. *P < 0.04, when Tuj1-positive axons in Poly(I:C)-treated eyes were compared with PBS, PBS plus control RNA, and PBS plus Rtca siRNA-treated eyes. **P < 0.03, when Tju1-positive axons in Poly(I:C) plus Rtca siRNA-treated eyes were compared with Tuj1-positive axons in Poly(I:C) or Poly(I:C) plus control RNA-treated eyes. 
Rtca siRNA Attenuates Poly(I:C)-Mediated Axonal Loss in the Optic Nerves of C57BL/6J Mice
Finally, to determine whether Rtca siRNA attenuates TLR3-mediated axonal loss in the optic nerves, C57BL/6J mouse eyes were treated with intravitreal injections of control RNA or Rtca siRNA, with or without PBS or Poly(I:C). At 24 hours after treatment, the axons were labeled with CTB and the animals were killed after an additional 24 hours. The results presented in Figures 9A and 9B indicate that when compared with the distance of CTB labeling in the optic nerves from PBS-treated eyes (580 ± 25 μm), the distance of CTB labeling in the optic nerves isolated from Poly(I:C)-treated eyes was decreased to 125 ± 4.04 μm. The distance of CTB labeling remained similar in optic nerves isolated from Poly(I:C) plus control RNA-treated mice. In contrast, the distance of CTB labeling was significantly increased in the optic nerves isolated from Poly(I:C) plus Rtca siRNA-treated eyes (550 ± 50 μm) when compared with Poly(I:C)-treated eyes. *P < 0.03, when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS, PBS plus control RNA, and PBS plus Rtca siRNA-treated eyes. **P < 0.04, when CTB-labeling in the optic nerves isolated from Poly(I:C) plus Rtca siRNA-treated eyes was compared with Poly(I:C) and Poly(I:C) plus control RNA-treated eyes.  
Figure 9
 
Inhibition of axonal loss in optic nerves of Rtca siRNA-treated C57BL/6J mice. C57BL/6J mouse eyes were treated with intravitreal injections of control RNA or Rtca siRNA, with or without PBS and Poly(I:C). At 24 hours after the treatments, axons were labeled with CTB and the animals were killed. CTB labeling in radial sections of optic nerves was observed under a fluorescence microscope (A), and the distance of CTB labeling was quantified by using NIH ImageJ software (B). *P < 0.03 when CTB labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.04 when CTB labeling in Poly(I:C) plus Rtca siRNA-treated eyes was compared with Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Figure 9
 
Inhibition of axonal loss in optic nerves of Rtca siRNA-treated C57BL/6J mice. C57BL/6J mouse eyes were treated with intravitreal injections of control RNA or Rtca siRNA, with or without PBS and Poly(I:C). At 24 hours after the treatments, axons were labeled with CTB and the animals were killed. CTB labeling in radial sections of optic nerves was observed under a fluorescence microscope (A), and the distance of CTB labeling was quantified by using NIH ImageJ software (B). *P < 0.03 when CTB labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.04 when CTB labeling in Poly(I:C) plus Rtca siRNA-treated eyes was compared with Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Discussion
Irreversible loss of RGCs and their axons leads to vision loss in POAG patients.1,2 Although elevated IOP has been identified as a risk factor for the loss of RGCs, the mechanisms that lead to the loss of RGCs and their axons in POAG are unclear. Although some previous studies have reported the role of oxidative stress in RGC loss in glaucoma,8,1518 the nature of the mediators and the cellular events that lead to oxidative stress-induced RGC loss are still unclear. A few previous studies have reported that TLRs play a role in RGC degeneration in POAG through glia-mediated neuro-inflammation or IOP-mediated oxidative stress.8,1823 For example, Luo et al.9 reported that TLR3 is expressed in astrocytes, and Toll-like receptor 2, TLR3, and Toll-like receptor 4 were expressed in microglia in the retinas obtained from human donor eyes affected with POAG.9 However, previous studies on the central nervous system (CNS) have reported that glial cells do not express TLR3,24 and a recent study from this laboratory reported that Poly(I:C) activated TLR3 in RGCs, but not in glial cells. Also, we have reported that the inhibition of TLR3 activation by a TLR3-specific inhibitor attenuated RGC loss to a large extent, but not completely, indicating that other proteins might play a role.10 
Interestingly, a recent study reported that elevated levels of Rtca promoted axonal loss following optic nerve crush in mice.11 Rtca regulates RNA splicing and repair mechanisms by converting the 3′-phosphate end of a spliced or damaged RNA into a 2′, 3′-cyclic phosphate. Recent studies have demonstrated that class IV dendritic arborization (da) neurons in the Drosophila peripheral nervous system were capable of regeneration, but not in the CNS.25 Using this model, Song et al.11 performed a candidate-gene approach primarily focusing on axotomy-regulated genes from various organisms and reported that Drosophila Rtca (CG401), a cellular RNA-processing enzyme of unknown function acts as an inhibitor of axonal regeneration in the CNS. Furthermore, Song et al. reported that axonal degeneration was inhibited in Rtca knockout mice following optic nerve crush, demonstrating that the degenerative role of Rtca was conserved in mammals. Therefore, by using a Poly(I:C)-induced mouse model of RGC degeneration that we reported previously,10 experiments in this study were designed to address the following two important questions related to axonal loss: (1) whether activation of TLR3 rather than a physical injury (crush) to the optic nerve promotes axonal loss and (2) whether activation of TLR3 promotes axonal loss by elevating Rtca protein levels in RGCs. 
The results presented in this study show that not only a physical injury to the optic nerve (crush)11 but also activation of the TLR3 promote axonal loss in the eye by up-regulating the protein levels of Rtca in RGCs. The results also demonstrate that the deletion of Tlr3 or the use of Rtca siRNA down-regulates Rtca protein levels in the retina and attenuates TLR3-mediated axonal loss. Interestingly, Rtca siRNA also decreased JNK3 and pJNK3 levels, indicating that JNK3 is a downstream target of Rtca. Taken together, these results suggest that the elevated levels of the Rtca protein play a major role in axonal loss in the retina and the optic nerve, regardless of the type of injury to the axons. If the role of Rtca in axonal degeneration can be confirmed in other animal models of retinal degeneration including POAG, targeted inhibition of Rtca may improve the treatment of axonopathies. 
Acknowledgments
Supported by grants from the Center for Biomedical Research and the University Research Committee of Oakland University to SKC. 
Disclosure: S.K. Chintala, None; N. Daram None 
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Figure 1
 
Loss of RGCs and their axons in CB57BL/6J mouse retinas. At 24, 48, and 72 hours after PBS or Poly(I:C)-treatment, RGC loss was determined by immunostaining of whole retinas with Brn3a antibody (A, leftmost panels) and axonal loss was determined with Tuj1 antibody (A, middle panels). After immunostaining, the number of remaining RGCs and axons in eight areas of equal size (B), located at an equal distance from the optic disc were quantified by using NIH ImageJ software. Bar graph C shows the quantification of remaining RGCs, and bar graph D shows the remaining axons at each time point. #P < 0.015 when Brn3a-positive RGCs in Poly(I:C)-treated retinas were compared with PBS-treated retinas. *P < 0.02 when Tuj1-positive axons in Poly(I:C)-treated retinas were compared with axons in PBS-treated retinas.
Figure 1
 
Loss of RGCs and their axons in CB57BL/6J mouse retinas. At 24, 48, and 72 hours after PBS or Poly(I:C)-treatment, RGC loss was determined by immunostaining of whole retinas with Brn3a antibody (A, leftmost panels) and axonal loss was determined with Tuj1 antibody (A, middle panels). After immunostaining, the number of remaining RGCs and axons in eight areas of equal size (B), located at an equal distance from the optic disc were quantified by using NIH ImageJ software. Bar graph C shows the quantification of remaining RGCs, and bar graph D shows the remaining axons at each time point. #P < 0.015 when Brn3a-positive RGCs in Poly(I:C)-treated retinas were compared with PBS-treated retinas. *P < 0.02 when Tuj1-positive axons in Poly(I:C)-treated retinas were compared with axons in PBS-treated retinas.
Figure 2
 
Axonal loss in the optic nerves of C57BL/6J mice. One day before euthanizing the mice that were treated with PBS or Poly(I:C), axons in the optic nerves were anterogradely labeled with AlexaFlour 555–conjugated CTB. At 24, 48, and 72 hours after the treatments, radial sections of the optic nerves were prepared and imaged by using a fluorescence microscope (A). Data presented in B show a progressive decrease of CTB-labeling in the optic nerves isolated from Poly(I:C)-treated eyes when compared with the optic nerves isolated from PBS-treated eyes. *P < 0.01 when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with CTB-labeling in the optic nerves from PBS-treated eyes.
Figure 2
 
Axonal loss in the optic nerves of C57BL/6J mice. One day before euthanizing the mice that were treated with PBS or Poly(I:C), axons in the optic nerves were anterogradely labeled with AlexaFlour 555–conjugated CTB. At 24, 48, and 72 hours after the treatments, radial sections of the optic nerves were prepared and imaged by using a fluorescence microscope (A). Data presented in B show a progressive decrease of CTB-labeling in the optic nerves isolated from Poly(I:C)-treated eyes when compared with the optic nerves isolated from PBS-treated eyes. *P < 0.01 when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with CTB-labeling in the optic nerves from PBS-treated eyes.
Figure 3
 
Up-regulation of Rtca protein in the retinas of C57BL/6J mice. (A) Retinal proteins extracted from PBS or Poly(I:C)-treated eyes (50 μg) were subjected to Western blot analysis by using antibodies for Rtca and TLR3. Actin antibody was used to determine equal loading of the proteins. (B) The bar graph shows the relative levels of Rtca and TLR3 in retinal protein extracts. Results presented in the figure show that Poly(I:C) up-regulated the protein levels of both Rtca and TLR3 in the retina. *P < 0.03 when TLR3 and Rtca protein levels in Poly(I:C)-treated retinas were compared with TLR3 and Rtca protein levels in PBS-treated retinas.
Figure 3
 
Up-regulation of Rtca protein in the retinas of C57BL/6J mice. (A) Retinal proteins extracted from PBS or Poly(I:C)-treated eyes (50 μg) were subjected to Western blot analysis by using antibodies for Rtca and TLR3. Actin antibody was used to determine equal loading of the proteins. (B) The bar graph shows the relative levels of Rtca and TLR3 in retinal protein extracts. Results presented in the figure show that Poly(I:C) up-regulated the protein levels of both Rtca and TLR3 in the retina. *P < 0.03 when TLR3 and Rtca protein levels in Poly(I:C)-treated retinas were compared with TLR3 and Rtca protein levels in PBS-treated retinas.
Figure 4
 
Localization of Rtca protein in RGCs of C57BL/6J mice. At 24, 48, and 72 hours after PBS or Poly(I:C)-treatment, retinal cross-sections prepared from C57BL/6J mouse eyes were immunostained by using an antibody against Rtca and double labeled with Tuj1 antibody. Results presented in the figure show that Rtca protein, absent in cross-sections prepared from PBS-treated eyes, was elevated in retinal cross-sections prepared from Poly(I:C)-treated eyes and colocalized in Tuj1-positive RGCs (red arrows). The white bar indicates a size of 50 μm.
Figure 4
 
Localization of Rtca protein in RGCs of C57BL/6J mice. At 24, 48, and 72 hours after PBS or Poly(I:C)-treatment, retinal cross-sections prepared from C57BL/6J mouse eyes were immunostained by using an antibody against Rtca and double labeled with Tuj1 antibody. Results presented in the figure show that Rtca protein, absent in cross-sections prepared from PBS-treated eyes, was elevated in retinal cross-sections prepared from Poly(I:C)-treated eyes and colocalized in Tuj1-positive RGCs (red arrows). The white bar indicates a size of 50 μm.
Figure 5
 
Rtca, JNK3, and pJNK3 levels were not up-regulated in Tlr3 knockout mice. At 48 hours after treating C57BL/6J and Tlr3 knockout mouse eyes with PBS or Poly(I:C), Western blot analysis was performed on total proteins extracted from the retinas by using Rtca, JNK3, and pJNK3 antibodies (A). Actin bands show the loading of an equal amount of total proteins. The bar graph in B shows semiquantitative analysis of Rtca, JNK3, and pJNK3 levels from the Western blots. *P < 0.02 when Rtca, JNK3, and pJNK3 levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes were compared with Rtca, JNK3, and pJNK3 in retinal proteins extracted from PBS-treated C57BL/6J mouse eyes. **P < 0.03 when Rtca, JNK3, and pJNK3 levels in retinal protein extracted from Poly(I:C)-treated Tlr3 knockout mice were compared with Poly(I:C)-treated C57BL/6J mouse eyes.
Figure 5
 
Rtca, JNK3, and pJNK3 levels were not up-regulated in Tlr3 knockout mice. At 48 hours after treating C57BL/6J and Tlr3 knockout mouse eyes with PBS or Poly(I:C), Western blot analysis was performed on total proteins extracted from the retinas by using Rtca, JNK3, and pJNK3 antibodies (A). Actin bands show the loading of an equal amount of total proteins. The bar graph in B shows semiquantitative analysis of Rtca, JNK3, and pJNK3 levels from the Western blots. *P < 0.02 when Rtca, JNK3, and pJNK3 levels in retinal proteins extracted from Poly(I:C)-treated C57BL/6J mouse eyes were compared with Rtca, JNK3, and pJNK3 in retinal proteins extracted from PBS-treated C57BL/6J mouse eyes. **P < 0.03 when Rtca, JNK3, and pJNK3 levels in retinal protein extracted from Poly(I:C)-treated Tlr3 knockout mice were compared with Poly(I:C)-treated C57BL/6J mouse eyes.
Figure 6
 
Loss of RGCs and their axons is attenuated in Tlr3 knockout mouse retinas. At 48 hours after treating Tlr3 knockout mouse eyes with PBS or Poly(I:C), axonal loss in the retina was determined by immunostaining of whole retinas with Tuj1 antibody. The results presented in A and B indicate that Poly(I:C) did not promote axonal loss in Tlr3 knockout mouse retinas when compared with the axons in PBS-treated Tlr3 knockout mouse eyes. NS, not significant when the number of axons in Poly(I:C)-treated retinas was compared with PBS-treated retinas. To determine axonal loss in the optic nerves, 24 hours before euthanizing PBS or Poly(I:C)-treated Tlr3 knockout mice, axons in the optic nerves were anterogradely labeled with CTB. At 24 hours after CTB labeling, mice were euthanized, radial sections of the optic nerves were prepared, and CTB-labeling in optic nerves was assessed by observing them under a fluorescence microscope (C). Results presented in C and D indicate that Poly(I:C) did not promote axonal loss in the optic nerves from Tlr3 knockout mice. NS, not significant when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS-treated eyes.
Figure 6
 
Loss of RGCs and their axons is attenuated in Tlr3 knockout mouse retinas. At 48 hours after treating Tlr3 knockout mouse eyes with PBS or Poly(I:C), axonal loss in the retina was determined by immunostaining of whole retinas with Tuj1 antibody. The results presented in A and B indicate that Poly(I:C) did not promote axonal loss in Tlr3 knockout mouse retinas when compared with the axons in PBS-treated Tlr3 knockout mouse eyes. NS, not significant when the number of axons in Poly(I:C)-treated retinas was compared with PBS-treated retinas. To determine axonal loss in the optic nerves, 24 hours before euthanizing PBS or Poly(I:C)-treated Tlr3 knockout mice, axons in the optic nerves were anterogradely labeled with CTB. At 24 hours after CTB labeling, mice were euthanized, radial sections of the optic nerves were prepared, and CTB-labeling in optic nerves was assessed by observing them under a fluorescence microscope (C). Results presented in C and D indicate that Poly(I:C) did not promote axonal loss in the optic nerves from Tlr3 knockout mice. NS, not significant when CTB-labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS-treated eyes.
Figure 7
 
Down-regulation of Rtca, JNK3, and pJNK3 in the retinas from Rtca siRNA-treated C57BL/6J mice. Equal amounts of retinal proteins (50 μg) extracted from the eyes treated with control RNA or Rtca siRNA, with or without PBS or Poly(I:C) were subjected to Western blot analysis by using antibodies for Rtca, JNK3, and pJNK3 (A). Actin bands indicate loading controls for the total proteins. The bar graph (B) shows the relative levels of Rtca, JNK3, and pJNK3 in retinal protein extracts. *P < 0.03 and #P < 0.04 when Rtca, JNK3, and pJNK3 levels in the retinal proteins extracted from Poly(I:C)-treated eyes were compared with Rtca, JNK3, and pJNK3 levels in the retinal proteins extracted from PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.02 when Rtca, JNK3, and pJNK3 levels in retinal proteins extracted from Poly(I:C) plus Rtca siRNA-treated eyes were compared with Rtca, JNK3, and pJNK3 levels in Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Figure 7
 
Down-regulation of Rtca, JNK3, and pJNK3 in the retinas from Rtca siRNA-treated C57BL/6J mice. Equal amounts of retinal proteins (50 μg) extracted from the eyes treated with control RNA or Rtca siRNA, with or without PBS or Poly(I:C) were subjected to Western blot analysis by using antibodies for Rtca, JNK3, and pJNK3 (A). Actin bands indicate loading controls for the total proteins. The bar graph (B) shows the relative levels of Rtca, JNK3, and pJNK3 in retinal protein extracts. *P < 0.03 and #P < 0.04 when Rtca, JNK3, and pJNK3 levels in the retinal proteins extracted from Poly(I:C)-treated eyes were compared with Rtca, JNK3, and pJNK3 levels in the retinal proteins extracted from PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.02 when Rtca, JNK3, and pJNK3 levels in retinal proteins extracted from Poly(I:C) plus Rtca siRNA-treated eyes were compared with Rtca, JNK3, and pJNK3 levels in Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Figure 8
 
Inhibition of RGC and axonal loss in the retinas of Rtca siRNA-treated C57BL/6J mice. At 48 hours after treating C57BL/6J mouse eyes with control RNA or Rtca siRNA, with or without PBS or Poly(I:C), RGC loss was determined by immunostaining whole retinas with Brn3a antibody (A, left panels) and axonal loss was determined with Tuj1 antibody (A, middle panels). After immunostaining, the number of remaining RGCs (B) and their axons (C) in the retinas was quantified by using NIH ImageJ software. #P < 0.04, when the number of Brn3a-positive RGCs in Poly(I:C)-treated retinas was compared with PBS-treated retinas. ##P < 0.03 when the number of Brn3a-positive RGCs in Poly(I:C) plus Rtca siRNA-treated retinas was compared with Poly(I:C)-treated retinas. *P < 0.04 when Tuj1-positive axons in Poly(I:C)-treated eyes were compared with PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.03 when Tuj1-positive axons in Poly(I:C) plus Rtca siRNA-treated eyes were compared with Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Figure 8
 
Inhibition of RGC and axonal loss in the retinas of Rtca siRNA-treated C57BL/6J mice. At 48 hours after treating C57BL/6J mouse eyes with control RNA or Rtca siRNA, with or without PBS or Poly(I:C), RGC loss was determined by immunostaining whole retinas with Brn3a antibody (A, left panels) and axonal loss was determined with Tuj1 antibody (A, middle panels). After immunostaining, the number of remaining RGCs (B) and their axons (C) in the retinas was quantified by using NIH ImageJ software. #P < 0.04, when the number of Brn3a-positive RGCs in Poly(I:C)-treated retinas was compared with PBS-treated retinas. ##P < 0.03 when the number of Brn3a-positive RGCs in Poly(I:C) plus Rtca siRNA-treated retinas was compared with Poly(I:C)-treated retinas. *P < 0.04 when Tuj1-positive axons in Poly(I:C)-treated eyes were compared with PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.03 when Tuj1-positive axons in Poly(I:C) plus Rtca siRNA-treated eyes were compared with Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Figure 9
 
Inhibition of axonal loss in optic nerves of Rtca siRNA-treated C57BL/6J mice. C57BL/6J mouse eyes were treated with intravitreal injections of control RNA or Rtca siRNA, with or without PBS and Poly(I:C). At 24 hours after the treatments, axons were labeled with CTB and the animals were killed. CTB labeling in radial sections of optic nerves was observed under a fluorescence microscope (A), and the distance of CTB labeling was quantified by using NIH ImageJ software (B). *P < 0.03 when CTB labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.04 when CTB labeling in Poly(I:C) plus Rtca siRNA-treated eyes was compared with Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
Figure 9
 
Inhibition of axonal loss in optic nerves of Rtca siRNA-treated C57BL/6J mice. C57BL/6J mouse eyes were treated with intravitreal injections of control RNA or Rtca siRNA, with or without PBS and Poly(I:C). At 24 hours after the treatments, axons were labeled with CTB and the animals were killed. CTB labeling in radial sections of optic nerves was observed under a fluorescence microscope (A), and the distance of CTB labeling was quantified by using NIH ImageJ software (B). *P < 0.03 when CTB labeling in the optic nerves from Poly(I:C)-treated eyes was compared with PBS, PBS plus Rtca siRNA, and PBS plus control RNA-treated eyes. **P < 0.04 when CTB labeling in Poly(I:C) plus Rtca siRNA-treated eyes was compared with Poly(I:C) or Poly(I:C) plus control RNA-treated eyes.
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