|Molecular Vision 2006;
Received 17 February 2005 | Accepted 11 November 2005 | Published 22 February 2006
A selective method for transfection of retinal ganglion cells by retrograde transfer of antisense oligonucleotides against kynurenine aminotransferase II
Sebastian Thaler,1 Robert
Rejdak,1,2,3 Karen Dietrich,1 Thomas Ladewig,1 Etsuo
Okuno,4 Tomasz Kocki,5 Waldemar Andrzej Turski,5
Anselm Junemann,6,7 Eberhart Zrenner,1 Frank
1Department of Pathophysiology of Vision and Neuro-Ophthalmology, University Eye Hospital, Tübingen, Germany; 2First Eye Hospital and 5Department of Pharmacology, Medical University, Lublin, Poland; 3Medical Research Center, Polish Academy of Science, Warsaw, Poland; 4Department of Clinical Nutrition, Kyushu Nutrition Welfare University, Fukuoka, Japan; 6Department of Ophthalmology and 7University Eye Hospital, Friedrich-Alexander University Erlangen-Nurnberg, Erlangen, Germany
Correspondence to: Sebastian Thaler, Department of Pathophysiology of Vision and Neuro-Ophthalmology, University Eye Hospital, Röntgenweg 11, 72076 Tübingen, Germany; Phone: +49 7071 29 84781; FAX: +49 7071 29 5777; email: firstname.lastname@example.org
Purpose: Intravitreal administration of specific antisense oligonucleotides (ODNs) effectively downregulates gene expression in the retina but does not modulate it exclusively in retinal ganglion cells (RGCs). Expression of kynurenine aminotransferase II (KAT II) in RGCs has been well described in the literature. We describe a new method for downregulating cellular KAT II expression via transfection of RGC by retrograde transfer of ODN.
Methods: Fluorescently labeled, specific ODNs against KAT II were injected into rats either intravitreally or into the superior colliculi. Fluorescence microscopy of retinal flat-mounts and radial sections was used to compare the location, duration, and degree of transfection for both methods of delivery. The effects of both methods on KAT II expression in RGCs were studied immunohistochemically with unlabeled ODN. Retinal kynurenic acid (KYNA) contents were measured using high pressure liquid chromatography (HPLC).
Results: After intravitreal injection, fluorescently labeled ODN reached all retinal layers, whereas injections into the superior colliculus resulted in transfection of the RGC layer alone. Immunohistochemistry showed that both methods of ODN application had a similar effect on downregulation of KAT II expression in RGC. Retinal KYNA content decreased significantly 4 days after both types of ODN administration.
Conclusions: This study demonstrated that retrograde transfer of specific ODN into RGC is feasible and induces downregulation of KAT II cellular expression. This may become a useful tool for modulating gene expression in the retinal ganglion cell layer in vivo without direct transfer of ODN to other retinal cell layers.
Antisense oligonucleotides (ODNs) have been used in various tissues, including retinal tissue, as therapeutic agents, and as research tools for downregulating certain genes. The stability of phosphorothioate-backbone ODNs and their effectiveness in downregulating certain target genes have been well described. Phosphorothioate-backbone ODNs are frequently used for in vivo experimentation and for therapeutic purposes due to their improved stability compared to unmodified phosphodiester ODNs [1-5].
ODNs can be successfully introduced into the retina both intravitreally and via subretinal injection [1,6,7]. However, intravitreal injections have disadvantages and risks, including lens injury, modification of certain neuroprotective pathways, hemorrhage, infection, and undesired transfer of ODNs to nearly all retinal cell layers. These disadvantages indicate that there is a need to evaluate new methods of ODN delivery to retinal ganglion cells (RGCs). It has been shown that ODNs can be transported retrogradely into peripheral nerve cells with a simultaneous downregulating effect . Furthermore, it has been demonstrated in the rat brain that ODNs apparently penetrate cell membranes and accumulate in both the cytoplasm and the nucleus . In the present study we therefore used retrograde transport of ODNs to transfect RGCs.
Kynurenine aminotransferase II (KAT II; also named aminoadipate aminotransferase) is an enzyme expressed in the inner retina . In the central nervous system, KAT II is responsible for the formation of kynurenic acid (KYNA), the only known endogenous neuroprotective antagonist of ionotropic glutamate receptors  and α7 nicotinic acetylcholine receptors . Importantly, indications have been found that changes of KAT II expression and retinal KYNA content are involved in mechanisms of retinal ontogeny and degeneration [13-15].
The present study was the first to test ODN delivery to RGC using retrograde transport via injection into the superior colliculus. Since the presence of KAT II in RGCs is well documented [10,16], this study also compared the effects of intravitreal and retrograde ODN delivery on KAT II expression in RGC.
All experiments were performed in compliance with guidelines for animal care in the European Community and those of the Association for Research in Vision and Ophthalmology. Brown Norway rats (Charles River, Wilmington, MA) two months of age with a body weight of 150-200 g were used in the study. The animals were housed under a 12 h light-dark cycle with food and water ad libitum.
The phosphorothioate-backbone ODNs used in this study were designed to be 20 bases in length (complementary to the area of the start codon of KAT II mRNA) and commercially synthesized (biomers.net; Ulm, Germany). The nucleotide sequence for KAT II mRNA was obtained from the GenBank data base (GenBank accession number NM_017193).
The ODN sequence of KAT II-ODN was 5'-TTC ATG TCT CTG CTG GTC GC-3'. A scrambled KAT II-ODN was used as a control with the randomized sequence 5'-GTA CGT CTG TTC CTG TTC CG-3', a sequence showing no homology to known genes in the Rattus norvegicus genome.
Both Cy3-labeled and unlabeled ODN against KAT II, along with scrambled ODN or Cy3 fluorescent dye alone as controls, were dissolved in H2O (pH 7.4) at a final concentration of 100 μM for intravitreal injection and injection into the superior colliculus of rats. To determine dose-dependent effects on retinal KYNA content, experiments with unlabeled ODN at 10 μM, 50 μM, or 100 μM were performed.
Rats were anesthetized with an intraperitoneal injection of chloral hydrate (7%, 6 ml/kg body weight). Intravitreal ODN injections were carried out in each case with a heat-pulled glass capillary connected to a microsyringe (Drummond Scientific Co., Broomall, PA) under direct observation through a microscope. Animals with visible lens damage were excluded from the experiments and not used thereafter. A single injection of 2 μl of 10 μM, 50 μM, and 100 μM (200 pmol) ODN was given. In each case, the contralateral eye served as a control eye and was injected with the scrambled ODNs.
The animals were sacrificed with CO2 1 day, 3 days, 4 days, 7 days, and 14 days after injection, and the eyes were enucleated immediately. Groups of 3-4 animals were used for each setup to ensure reproducibility of results. Following hemisection of the eyes along the ora serrata, the cornea, lens, and vitreous body were removed. Eyecups were immersion-fixed for 30 min in 4% (w/v) paraformaldehyde (PFA) in phosphate buffer (PB; 0.1 M Na2HPO4, pH 7.4) at 4 °C. After being washed 3 times in PB, tissues were cryoprotected by immersion in 30% (w/v) sucrose in PB overnight at 4 °C. Samples were then embedded in cryomatrix (Jung, Leica Microsystems Nussloch GmbH; Nussloch, Germany). The embedded eye cups were cut radially in 10 to 12 μm sections using a cryostat, collected on silane-coated slides, air dried, and stored at -20 °C for further use.
Sections were washed with PBS for 5 min. Endogenous peroxidase was blocked with 3% H2O2 in 40% methanol. Retinal sections were then incubated in a solution containing 10% normal goat serum and 0.3% PBST (0.05 M Na2HPO4, pH 7.4; 0.3% Triton X-100) to reduce background staining. The primary antibodies were diluted 1:100 in PBST containing 10% NGS and applied overnight at 4 °C. A rabbit polyclonal antibody against KAT II was used to detect KAT II immunoreactivity. The specificity of this antibody was tested in previous studies [17,18]. The next day, the sections were rinsed with PBS (three times for 5 min each) followed by incubation with a biotin-conjugated goat anti-rabbit IgG secondary antibody (1:200; Vectastain Elite Kit, Vector Laboratories, Burlingame, CA) in PBST containing 5% NGS for 1 h at room temperature. After being rinsed in PBS, sections were processed with an avidin-biotin-peroxidase complex for 30 min at room temperature (Vectastain Elite Kit) and stained with diaminobenzidine as the chromophore. For the control sections the primary antibody was omitted. Sections were viewed using an Olympus AX70 microscope (Olympus Optical Co. GmbH; Hamburg, Germany).
Labeling was performed either with a fluorescent tracer or with ODN. Animals were anesthetized deeply, and a total of 7 μl fluorescence-labeled ODNs (100 μM) or unlabeled ODN (10 μM, 50 μM, or 100 μM) was applied to each superior colliculus by three stereotaxic injections. For RGC-labeling, 7 μl of the fluorescent tracer hydroxystilbamidine methanesulfonate (Fluorogold, Molecular Probes, Eugene, OR) was applied accordingly. In some cases a combination of Fluorogold and fluorescence-labeled ODN was applied. The animals were sacrificed with CO2 1 day, 3 days, 4 days, 7 days, or 14 days later. Groups of 3-4 animals were used for each setup to ensure reproducibility of results. The eyes were enucleated, the retinas dissected, flat-mounted on cellulose nitrate filters (pore size 60 μm; Sartorius, Long Island, NY), and fixed in 2% PFA for 30 min. Observation was performed under a fluorescence microscope (Olympus AX70) immediately. Images were obtained via a digital imaging system connected to the microscope (analySIS 3.2; Soft Imaging System GmbH, Münster, Germany). For pictures taken with monochromatic excitation, a polychrome monochromator (Till Photonics GmbH, Martinsried, Germany) was used, images were obtained via a digital imaging system (TillVision 4.0, Till Photonics GmbH, Martinsried, Germany).
Analysis of KYNA
Retinal KYNA content was measured 4 days after ODN injections. The animals were killed with CO2, and the eyes were removed. To obtain isolated neural retinas, the eyes were opened along the ora serrata and cornea, and the lens and vitreous body were removed. Using a pair of forceps, the whole neural retinas were then carefully dissected free from the retinal pigment epithelium and sclera. Retinas were immediately frozen in liquid nitrogen after removal.
High pressure liquid chromatography
KYNA levels were investigated according to the method of Turski and colleagues . Specimens were sonicated in 2 volumes (w/v) of distilled water, immersed in a boiling water bath for 10 min, and centrifuged (10 min, 18,500x g). The resulting supernatant was diluted (1:1) with 0.2 N HCl and applied to a Dowex 50-W column, hydrogen form, prewashed with 0.1 N HCl. Columns were subsequently washed with 1 ml 0.1 N HCl and 1 ml water. KYNA was eluted with 2 ml of water. The elute was subjected to HPLC, and KYNA was detected fluorimetrically according to the method of Shibata . HPLC reagents used in the study were obtained from Baker (Griesheim, Germany) and were of the highest available purity. Statistical analysis was performed using the unpaired Student's t-test.
Injection of Cy3-labeled oligonucleotides
Fluorescence microscopy was used to study transfection of the retina, and RGCs in retinal flat-mounts, and radial sections 1 day to 2 weeks after intravitreal injection of Cy3-labeled ODN or retrograde injection into the superior colliculus.
Fluorescence-labeled RGCs were studied in retinal flat-mounts 1 day (Figure 1A), 3 days (Figure 1C), 7 days (Figure 1E), and two weeks (Figure 1G) after retrograde injection of Cy3-labeled ODN into the superior colliculus of rats. Staining remained strong over the whole period of time and showed a homogeneous pattern in the whole retina. Fluorescence did not appear in retinal layers other than the retinal ganglion cell layer when detected at times from 1 day to two weeks thereafter in radial sections of the retina (Figure 1I-L). Control injections of fluorescent Cy3-NHS-ester alone (i.e., not coupled with ODN) into the superior colliculus produced no detectable fluorescence whatever in the retina (data not shown).
Fluorescently labeled cells were detected in the RGC layer in retinal flat-mounts 1 day (Figure 1B), 3 days (Figure 1D), 7 days (Figure 1F), and two weeks (Figure 1H) after intravitreal injection of Cy3-labeled ODNs into rats. The staining showed a homogeneous pattern in the whole retina. Radial sections revealed fluorescently labeled cells in other layers of the retina, even staining in the outer nuclear layer after 3 days (Figure 1M-P). This pattern remained similar over the whole time period (other times not shown).
Laser scanning microscopy
Laser scanning microscopy revealed fluorescent ODNs in the cytoplasm and in the nucleus of RGCs three days after both types of application. Following injection into the superior colliculus, however, only cells in the ganglion cell layer were transfected with ODN (red staining, Figure 2A). After intravitreal injections, both RGC (red) and other layers of the retina were transfected with ODNs (blue staining, Figure 2B).
Expression of KAT II in RGC was studied at different times up to one week after injection of unlabeled, specific ODN (100 μM; Figure 3). Downregulation of KAT II expression was visible 1 day after injection (Figure 3D) and reached a maximum 3 days after injection into the superior colliculus (Figure 3E). Seven days after injection, KAT II staining was stronger again (Figure 3F). The same was true following intravitreal injection (Figure 3J-L). Staining with the scrambled ODN controls (Figure 3C,I) was similar to that in untreated rats (Figure 3G).
Costaining of RGCs with fluorescently labeled ODN and Fluorogold
Following injections both intravitreally and into the superior colliculus combined with retrograde labeling of RGC with Fluorogold (FG), images were recorded using fluorescence microscopy under monochromatic excitation of Cy3 (450 nm) and FG (360 nm, Figure 4).
After Cy3-ODN injections into the superior colliculus and labeling of RGC with FG, all RGCs labeled with FG (Figure 4B) were also stained with Cy3 (Figure 4A). No cells other than FG-marked RGCs showed Cy3-positive staining. This pattern remained similar when measured at times from 3 days to 2 months thereafter (data not shown). Cy3-fluorescence was strongest three days after injection and clearly weaker two weeks after injection. In contrast, FG-fluorescence remained at roughly the same intensity over the whole period of time (data not shown). After intravitreal injection of Cy3-ODN and labeling of RGC with FG, all RGC labeled with FG (Figure 4D) were also stained with Cy3 (Figure 4C). Additionally, some cells other than the FG-labeled RGC were stained with Cy3 (compare Figure 4C,D). This pattern remained similar for up to two months after injection (data not shown).
High pressure liquid chromatography
Concentrations of KYNA measured in retinas of eyes injected with scrambled ODN (intravitreally injected: 103.1±7.1 pmol/g wet weight, mean±standard error, n=11; retrogradely administered: 96±2.9 pmol/g wet weight, n=12) were similar to those observed in a previous study in untreated rat eyes (99.9±24.6 pmol/g wet weight, n=8) . The efficacy of different ODN concentrations was tested in order to elucidate the most effective dose (Figure 5). We observed a concentration of 10 μM not to influence retinal KYNA content (intravitreally injected: 101.5±2.1 pmol/g wet weight, n=11; retrogradely administered: 93.6±2.1 pmol/g wet weight, n=10). After administration of ODN at a concentration of 50 μM both retrogradely (84.2±5.4 pmol/g wet weight, n=10) and intravitreally (86.1±4.5 pmol/g wet weight, n=10) a tendency toward a decrease in KYNA concentration was observed yet a significant change was not found (p=0.0588 and 0.0623, respectively). However intravitreal ODN injection at a concentration of 100 μM induced a significant (p<0.01) decrease of retinal KYNA content (67.04±12 pmol/g wet weight, n=8). Retrograde administration of 100 μM ODN led to an even stronger decrease (p<0.001) of retinal KYNA formation (48.440±3.090 pmol/g wet weight, n=11).
Our results show that RGCs can be transfected retrogradely by means of ODN injections into the superior colliculus in rats. We found that this method, like intravitreal ODN administration, induces downregulation of KAT II expression in RGCs. We therefore suggest that the procedure described here may prove useful as a tool for modulation of other target genes in RGCs.
No fluorescence was detectable in the retina for up to 4 days after retrograde application of the fluorescent Cy3-NHS-ester alone, indicating that retrograde transfection of RGCs bodies did not result from the coupled fluorescent dye. Rather, transfection apparently took place only via retrograde transfer of ODN. After injection of fluorescently labeled ODN into the superior colliculus, only cells in the RGC layer were transfected. Colabeling experiments in this study show that the transfection efficiency of retrogradely labeled ODNs for RGCs is equal to fluorogold labeling, which is about 90-95%, as not all RGC in rat project to the superior colliculi . These results suggest that retrograde transfer can make it possible to study the effect of ODNs solely on RGCs in vivo. In this way, new insights can be gained into the role of certain target genes expressed in RGCs and the role of RGC in retinal function.
Ganglion cell gene modulation in vivo offers major advantages over most knock-out mice models, since downregulation of genes is not limited in the majority of knock-out mice to a specific tissue, cellular layer, or group of cells. Conditional knockout and knock-ins with Cre/Cox (Floxed) technology are viable options, however, if compared to ODN treatment, these models are much more complex and time consuming. ODN treatment in vivo also offers advantages over in vitro models of RGC cultures: RGC culture systems do not provide opportunities for studying how adjoining tissues or organ systems are affected by downregulation of genes via ODN or viral transfection. It has been shown that retrograde transport of viral vectors from the superior colliculus into the retina is feasible  and that viral vectors transported retrogradely in brain tissue through axonal transport to neuronal cell bodies can result in transgene expression [23,24]. In contrast to the delivery of siRNA to RGCs via in situ application to the proximal nerve stump after axotomy demonstrated by Lingor and colleagues , our method of delivering ODN retrogradely provides a way to interfere with gene expression under physiological conditions. Importantly, intervention via antisense treatment is feasible during physiological cell function and in different models of RGC damage. Our study revealed an intracellular localization of ODN with a distribution into the cytoplasm and nucleus after both methods of transfection. This agrees with previous findings that nuclear localization of ODN is frequently combined with a downregulating effect . While some studies have indicated that localization within the nucleus is an artifact of fixation , radioactive fractionation in another study showed that 15-20% of PS-ODN distribute themselves into the nucleus and 60% into cytosol in vascular smooth muscle cells . Our observations after both types of ODN delivery suggest that fluorescence in RGCs persists at least 14 days. In agreement with our findings, Shen et al.  used gene scan analysis to demonstrate persistence and integrity of PS-ODNs in the retina for at least 12 weeks after intravitreal injection.
Interestingly, we observed downregulation of KAT II expression for up to 7 days after both types of ODN administration. Studies performed with monkeys have indicated that retinal ODN levels induced with intravitreal injection remained similar for up to one week to those found to be effective in vitro [30,31]. Application of ODNs at consecutive times could result in a longer downregulating effect. This is currently being tested by our group. Data have been gathered showing penetration of ODN into RGCs, photoreceptors, and the retinal pigmented epithelium layer after intravitreal administration . Our experiments revealed similar results after intravitreal injection. Importantly, retrograde transfer of ODNs led to an accumulation of fluorescence solely in RGCs. Increased intraocular pressure poses a possible problem for retrograde transport of ODN. It has been suggested that retrograde axoplasmatic transport blockade is a major factor in RGC apoptosis [33,34]. Kim and colleagues  showed that retrograde axoplasmatic flow was significantly reduced by an increase in intraocular pressure. Moreover, it may not be possible to deliver ODNs to apoptotic cells. Further studies are required to show whether ODNs can be transported retrogradely to RGC in sufficient quantities when intraocular pressure is increased or other pathological conditions are present.
The ODN-induced downregulation of retinal KAT II found in the present study may have relevance for retinal function: Targeted deletion of the KAT II gene has recently revealed a critical role of its product, KYNA, in the regulation of synaptic transmission via α7 nicotinic receptors in the hippocampus . Whether this is also true for the retina remains to be determined. The present study also demonstrated that retinal content of KYNA decreased after both types of ODN administration in a dose-dependent manner. We observed a concentration of 100 μM to be the most effective. ODNs of this concentration induced a significant decrease of retinal KYNA content. KYNA concentrations measured in retinas of eyes injected with scrambled ODN as a control were similar to those in untreated rat eyes . Since retrograde ODN injection resulted in a stronger inhibition of KYNA formation, we speculate that this type of ODN delivery may induce more pronounced functional changes in RGC.
In conclusion, we found that retrograde transfer of specific ODN induces downregulation of KAT II expression in RGC, providing a new method of RGC transfection. This approach may facilitate investigations of retinal gene expression, architecture, and circuitry.
Supported by the Paul-Blümel-Stiftung; the Dr. Wolfbauer Stiftung; the Stiftung für Pathobiochemie und Molekulare Diagnostik and the EU (Marie Curie Individual Fellowship QLK2-CT-2002-51562; integrated project EVI-GENORET LSHG-CT-2005-512036). The authors thank Sylvia Bolz, Sandra Bernhard-Kurz, and Birgit Fehrenbacher for excellent technical assistance.
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