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. 2016 Feb;57(2):372-86.
doi: 10.1167/iovs.15-17703.

Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide

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Free PMC article

Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide

Cristhian J Ildefonso et al. Invest Ophthalmol Vis Sci. .
Free PMC article

Abstract

Purpose: Oxidative stress has been linked to several ocular diseases, initiating an inflammatory response that increases tissue injury. The Nrf2 transcription factor regulates expression of antioxidant genes and is tightly regulated by Kelch-Like ECH-Associated Protein 1 (Keap-1). We evaluate the antioxidant and anti-inflammatory properties of an adeno-associated virus (AAV) vector delivering an Nrf2-derived peptide that binds Keap-1.

Methods: The sequence of the Nrf2 peptide was fused to a cell-penetrating peptide (Tat-peptide) sequence (TatNrf2mer). The effects of lentiviral-delivered TatNrf2mer were studied in vitro. Transcript (quantitative [q] RT-PCR) and protein levels (ELISA and immunofluorescence) were quantified. Cell viability was measured by MTT and Cell Titer assays. The AAV vectors were packaged with the TatNrf2mer fused to secretable green fluorescent protein (GFP) under the control of the small chicken β actin promoter. The protective effects of this vector were evaluated in a model of RPE oxidative injury and in a mouse model of uveitis after intravitreal injection.

Results: Expression of TatNrf2mer peptide induced antioxidant gene expression, blocked IL-1β secretion, and protected cells from oxidative injury. In mice, TatNrf2mer expression partially protected photoreceptor function based on ERG responses and optical coherence tomography measurements in the sodium iodate (NaIO3) model. Furthermore, sGFP-TatNrf2mer expression decreased IL-1β and IL-6 in the NaIO3-treated mice, and resulted in a 54% decrease in the number of inflammatory cells in the vitreous body of the endotoxin-induced uveitis mouse model.

Conclusions: The intravitreally delivered AAV-TatNrf2mer has antioxidant and anti-inflammatory effects in widely-used models of ocular injury, suggesting it also could be useful in ocular diseases associated with oxidative stress and inflammasome activation.

Figures

Figure 1
Figure 1
The TatNrf2mer peptide induces antioxidant genes and protects cells against oxidative stress. (A) Map of the plasmid containing the TatNrf2mer sequence. A DNA sequence coding for the HIV-1 Tat peptide fused to the Nrf2 peptide was designed and codon optimized for expression in humans and mice. This sequence was cloned in a lentiviral vector plasmid fused to the puromycin resistance gene by a T2A self-cleaving peptide sequence. (B) Detection of TatNrf2mer mRNA in stably transfected ARPE-19 cells expressing TatNrf2mer. ARPE-19 cells transduced with lentiviral vectors delivering either TatNrf2mer-PuroR or PuroR were selected in puromycin. Total RNA was isolated from both stable cells, and a cDNA library was generated to detect the presence of TatNrf2mer by RT-PCR. Lentiviral plasmid containing the TatNrf2mer sequence was used as a positive control. (C) Constitutive expression of TatNrf2mer induces the expression of ARE genes. Quantitative RT-PCR detecting the GSTM1 and NqO1 mRNA was performed using the cDNA library described in (B), using β-actin as a control. ARPE-19 stably expressing TatNrf2mer had greater expression of ARE genes. Assays were performed in triplicate. (D) TatNrf2mer expression protects ARPE-19 cells from paraquat and H2O2 induced oxidative stress. Stably transfected ARPE-19 cells were incubated with 55, 166, or 500 μM paraquat for 48 hours or with 800 μM H2O2 for 6 hours to induce oxidative damage. Afterwards, cell viability was assessed with the Cell Titration or MTT assay, respectively. Assays were performed in triplicate. Values are reported as average ± SD.
Figure 2
Figure 2
Expression of TatNrf2mer increases the expression of endogenous Nrf2 in ARPE-19 cells. ARPE-19 cells stably expressing either TatNrf2mer-T2A-PuroR (TatNrf2mer) or T2A-PuroR were selected by the addition of puromycin. Stably transduced cells were stained with an antibody against the Nrf2 protein. Secondary antibody conjugated to Alexa Fluor 488 chromophore (green) was used to detect the presence or absence of the anti-Nrf2 antibody. DNA staining with DAPI (blue) was used as a counter stain. An isotype control antibody was included to detect any nonspecific binding of the secondary antibody. The fluorescence intensity was quantified using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and the corrected total cell fluorescence (CTCF), formula described on the bar graph on the right. Values are reported as average ± SD (n = 3 images).
Figure 3
Figure 3
A secretable TatNrf2mer induces the expression of ARE genes. (A) Two lentiviral vectors delivering a secretable GFP (sGFP) or a sGFP fused to the TatNrf2mer by a furin cleavage site (FCS) were designed. Both constructs were cloned in-frame with the 2A-puroR sequence of the lentiviral vector to generate fusion proteins. Plasmids were packaged as lentiviral vector particles. (B) Distribution of GFP and sGFP-TatNrf2mer in stably transfected HEK293T cells. HEK293T cells were transduced with lentiviral vectors delivering either GFP, sGFP, or sGFP-TatNrf2mer and were selected in the presence of puromycin. The expression of GFP was evaluated by fluorescence microscopy. The sGFP and sGFP-TatNrf2mer had a different pattern of cellular distribution than the GFP expressing cells. DAPI staining was performed as a counter stain. (C) Stable cells were grown in low protein medium for 3 days. The conditioned media were collected and cells were lysed. The presence of GFP fused molecules was determined by Western blot using antibody to GFP. Although the sGFP-TatNrf2mer fused protein is detected in the lysate, the only band detected in the medium corresponds to cleaved GFP, thus suggesting the secretion and cleavage of the sGFP-TatNrf2mer protein. (D) The conditioned media from sGFP-TatNrf2mer increased the expression of two ARE genes in ARPE-19 cells. ARPE-19 cells were incubated with the 3-day conditioned media of either sGFP or sGFP-TatNrf2mer for 18 hours. Total RNA was isolated from these cells and a cDNA library was generated. The relative expression of the two ARE genes GSTM1 and NqO1 was measured by qRT-PCR using β-actin as a control transcript. Values are reported as average ± SD (n = 3 biological replicates).
Figure 4
Figure 4
Expression of the sGFP-TatNrf2mer AAV vector in mice retinas is safe. (A) Map of the pTR-smCBA-sGFP-TatNrf2mer AAV vector. The coding sequence of the secretable and cell-penetrating peptide was cloned in an AAV plasmid containing the small CMV enhancer/chicken β-actin hybrid promoter which is constitutively active in most cells within the retina. (B) Expression of sGFP-TatNrf2mer gene within the retina. C57BL/6J mice were intravitreally injected with 3 × 109 vector genome copies (vgc) of AAV2-QUAD(Y-F)-T497V vector delivering either GFP or sGFP-TatNrf2mer. Three weeks after vector injection, gene expression was detected by fluorescence funduscopy. Representative fundus images demonstrate that, in contrast to the localized fluorescence expression of GFP, eyes injected with sGFP-TatNrf2mer showed a diffused fluorescence consistent with the secretion of GFP. (C) Two weeks after viral vector injection, mice were evaluated by SD-OCT to detect changes within the different layers of the retina. Representative images of retinas transduced with AAV vectors delivering GFP (top) or sGFP-TatNrf2mer (bottom). (D) Quantification of the different retina layers. The thickness of each layer was measured using the segmentation analysis of Diver 2.0 software from Bioptigen. No statistically significant difference was observed in any layer between eyes injected with GFP or sGFP-TatNrf2mer vector. (E) TatNrf2mer expression within the retina was not deleterious to the electrophysiological activity of the tissue. Injected mice were evaluated by ERG to detect any differences in amplitude between the expression of GFP and sGFP-TatNrf2mer. Eyes injected with the sGFP-TatNrf2mer AAV vector had statistically significant higher a-wave and c-wave amplitudes (P < 0.05) when compared to eyes injected with the GFP AAV vector. ERG b-wave amplitudes also were elevated but did not reach statistical significance (P = 0.062). Values are reported as average ± SEM (n = 10 mice).
Figure 5
Figure 5
Gene delivery of TatNrf2mer increases the expression of antioxidant genes within the retina. C57BL/6J mice were injected intravitreally with 3 × 109 vgc/eye of AAV2-QUAD(Y-F) T497V delivering either GFP or sGFP-TatNrf2mer. One month after the injection, mice were euthanized, their retinas were harvested, and RNA isolated from them. A cDNA library from each treatment was prepared and qRT-PCR was performed to measure the relative levels of the ARE genes HO-1 and GSTM1 using β-actin as a control. Eyes treated with the sGFP-TatNrf2mer vector had higher levels of HO-1, GSMT1, NqO1, and Catalase (all antioxidant genes) when compared to GFP injected control eyes. Values are reported as average ± SEM (n = 5 biological samples).
Figure 6
Figure 6
Expression of sGFP-TatNrf2mer partially protects the electrical activity of the retina from acute oxidative damage of NaIO3. C57BL/6J mice were injected with the AAV vectors described in previous figures. One month after the intravitreal injection of the vector, mice were injected intraperitoneally with 35 mg/kg sodium iodate to induce oxidative stress within the RPE. Seven days later mice were evaluated by ERG. One week after NaIO3 injury, eyes injected with the sGFP-TatNrf2mer had partial protection of the ERG a-wave (A) and b-wave (B) but not c-wave (P = 0.20; [C]), when compared to GFP injected eyes. (DF) A second group of mice treated with the same AAV vectors received NaIO3 at a lower dose (25 mg/kg) and evaluated as in the first cohort. At this lower dose, the amplitude of all the waves was significantly higher among the eyes treated with the sGFP-TatNrf2mer. Histograms report the maximum amplitudes recorded at 0 dB using the UTAS BigShot Visual Diagnostic System (2.7 cd.s/m2). Values are reported as average ± SEM (n = 11 mice in first cohort, n = 9 mice in second cohort).
Figure 7
Figure 7
The secretable TatNrf2mer decreases inflammatory cytokines in the retinas of NaIO3 treated mice. Retinas and RPE from mice treated with 25 mg/kg NaIO3 were harvested and sonicated in PBS supplemented with protease inhibitors. The protein concentration in the cleared lysate was determined and diluted to 400 ng/mL protein. The concentration of IL-1β, IL-6, and MCP-1 were determined by ELISA using 10 μg total protein per sample. Biological samples were evaluated in triplicates. Values are reported as average ± SEM (n = 7 samples).
Figure 8
Figure 8
The secretable TatNrf2mer has anti-inflammatory properties in a mouse model of ocular inflammation. (A) ARPE-19 stably expressing PuroR (Vector) or TatNrf2mer-PuroR (TatNrf2mer) were incubated with or without 30 μM 4-hydroxynonenal (4-HNE) for 18 hours. The concentration of IL-1β in the conditioned media was quantified in triplicate by ELISA. (B) C57BL/6J mice were injected intravitreally with 3 × 109 vgc of AAV vector delivering either GFP or sGFP-TatNrf2mer (TatNrf2mer). One month later mice were injected intravitreally with 25 ng LPS and then were euthanized 24 hours later. Their eyes were harvested and analyzed by histology. Representative images of hematoxylin and eosin–stained sections of eyes injected with either GFP (top) or TatNrf2mer vectors are shown (bottom). (C) The number of cells within the vitreous of at least two sections per eye were quantified by two independent subjects who were not aware of the treatments. Eyes injected with the TatNrf2mer AAV vector had significantly lower numbers of infiltrating cells within the vitreous body than the eyes injected with the GFP AAV vector. Values are reported as average ± SEM (n = 2 biologic replicates in [A], n = 5 mice in [C]).
Figure 9
Figure 9
Mechanism of action of TatNrf2mer. The Nrf2mer peptide is derived from the Nrf2 gene domain that binds Keap-1 and penetrates cells when fused to the Tat peptide. Once inside the cell, the TatNrf2mer binds to Keap-1 liberating Nrf2, which can translocate into the nucleus where it induces the expression of antioxidant genes (e.g., NqO1 and HO-1). These genes then reduce the ROS within therefore protecting the cell from the deleterious effects of free radicals.

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