Abstract The lack of axonal regeneration is the major cause of vision loss after optic nerve injury in adult mammals. The deletion of the mTOR negative regulator phosphatase and tensin homolog PTEN enhances regeneration of adult corticospinal neurons and ganglion cells. We evaluated cell survival and axonal regeneration in a rat model of optic nerve axotomy. Compared with the wildtype AAV2 vector, the YF mutant AAV2 vector enhanced retinal ganglia cell survival and stimulated axonal regeneration to a greater extent 6 weeks after axotomy.
Taken together, our results demonstrate that PTEN knockdown with the YF AAV2 vector promotes retinal ganglion cell survival and stimulates long-distance axonal regeneration after optic nerve axotomy.
Therefore, the YF AAV2 vector might be a promising gene therapy tool for treating optic nerve injury. Neural Regen Res ; As a part of the central nervous system, the optic nerve in mammals has a very limited ability to regenerate its axons after injury, resulting in irreversible vision loss.
The failure of axonal regeneration has been attributed to the apoptosis of RGCs, insufficient intrinsic growth capacity of mature neurons, lack of suitable stimuli, and an inhibitory extracellular environment Moore and Goldberg, ; Fischer and Leibinger, Over the last few decades, numerous studies have shown that activation of the intrinsic growth capacity is able to induce a robust regenerative response in mature axotomized RGCs Goldberg, ; Yang and Yang, Deletion of phosphatase and tensin homolog PTEN , a negative regulator of mammalian target of rapamycin mTOR , has been demonstrated to enhance the regeneration of adult corticospinal neurons and RGCs Park et al.
However, conditional gene deletion cannot currently be translated to clinical practice, and therapies based on small-interfering RNA siRNA to knockdown the target gene may be potentially most useful for treatment of optic nerve diseases, such as glaucomatous optic neuropathy Guzman-Aranguez et al.
Fortunately, site-directed tyrosine Y -to-phenylalanine F mutation of capsid surface-exposed and highly conserved tyrosine residues has been reported to dramatically increase the transduction efficiency of self-complementary AAV2 following intraocular injection Petrs-Silva et al. Therefore, we hypothesized that Y-to-F mutated AAV2 might have great potential role in gene therapy for traumatic optic neuropathy. In the present study, we evaluated the intraocular transduction characteristics of intravitreally injected Y-to-F mutated AAV2.
SCXK and were used for all experiments. Briefly, in stage one, two polymerase chain reaction PCR extension reactions were performed in separate tubes for each mutant.
One tube contained the forward PCR primer and the other contained the reverse primer. In stage two, the two reactions were mixed and a standard PCR mutagenesis procedure was carried out.
PCR primers were designed to introduce changes from tyrosine to phenylalanine residues and a silent change to create a new restriction endonuclease site for screening purposes.
The solution was boiled for 5 minutes and then cooled to room temperature. The sequences of the shRNA primers were as follows in bold: The plasmid was sequenced to confirm identity. A robust and non-cell-specific CAG promoter controlled the expression of ZsGreen, acting as a reporter. Right long terminal repeat; L-LTR: Then, 72 hours after transfection, cells were harvested and AAV were purified by dialysis and virus gradient centrifugation in iodixanol.
Protein liquid chromatography was performed to obtain high-titer viral stocks. The viral titers were determined using quantitative PCR and normalized to 1. Briefly, the needle was inserted into the peripheral retina, just behind the ora serrata, and was carefully positioned to avoid damage.
Rats with traumatic cataract, retinal detachment, or vitreous hemorrhage were excluded from this study. Establishment of the optic nerve axotomy model Optic nerve axotomy of the right eye was performed as reported previously Koch et al.
In brief, the lateral canthus was incised along the orbital rim and the lacrimal gland was moved to the side. The eyeball was slightly rotated by pulling the superior rectus muscle. The optic nerve was then exposed intraorbitally, and crushed with jeweler's forceps Dumont 5; Roboz, Switzerland at a distance of at least 2 mm behind the eyeball for approximately 10 seconds, avoiding damage to the ophthalmic artery.
The vascular integrity of the retina was examined by fundoscopy. Rats in which the retinal vessel was injured were excluded from the study.
For quantifying the density of RGCs, whole retinas were dissected out. Frozen sections were blocked with immunostaining blocking buffer Beyotime, Shanghai, China and permeabilized with 0. Retinas were blocked with immunostaining blocking buffer and permeabilized with 0. The sections or retinas were rinsed with 0. After washing, the sections were examined under a fluorescence microscope Nikon Eclipse50i, Tokyo, Japan , and images were captured with a CCD camera.
The retinas immunostained with TUJ1 antibody were mounted onto pre-coated glass slides, and the images were captured under the fluorescence microscope. Sixteen fields in the mid portion of the retina approximately 0. Western blot assay Total retinal protein was extracted and quantified using a bicinchoninic acid protein assay kit Beyotime. Membranes were incubated with a rabbit anti-rat glutamate aspartate transporter GLAST monoclonal antibody 1: After washing in Tris-buffered saline with Tween, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody 1: The immune complexes were detected by enhanced chemiluminescence Millipore.
Each experiment was performed at least three times. At least five non-consecutive sections were examined under the fluorescence microscope for each animal. Statistical analysis was carried out using Stata One-way analysis of variance followed by the Bonferroni's post hoc test was used to compare multiple groups.
Pairwise comparison between groups was performed using Student's t-test. Results Efficiency of transgene expression of AAV2 vectors To evaluate the efficiency of transgene expression, Wt AAV2, single mutant, quadruple mutant and sextuple mutant vectors were injected into the intact eyes of rats containing normal populations of RGCs. The fluorescence intensity of GFP expression in the eyes injected intravitreally with the various vectors was initially analyzed on retinal flat mounts to quantitatively evaluate the transduction capacity for each vector 4 weeks after injection.
GFP expression in flat-mount retinas 4 weeks following intravitreal delivery. A—D Images of the posterior retina including the optic disc; E—H images of the peripheral retina. I The GFP signal intensity for the single mutant was higher than for all other vectors. Click here to view Assessment of AAV2 transgene expression To evaluate the transduction properties of vectors 4 weeks after intravitreal injection, immunohistochemistry was performed on retinal flat mounts and frozen sections.
Immunofluorescence in flat-mount whole retinas showing RGCs expressing the GFP transgene 4 weeks after intravitreal injection. Green fluorescent protein; RGCs: Click here to view Figure 4: Green fluorescent protein; GS: Substantial RGC loss was evident 6 weeks after optic nerve axotomy. G The number of surviving RGCs was decreased significantly 6 weeks after axotomy. Optic nerve crush; RGCs: Click here to view In addition to evaluating the survival of RGCs, we also assessed axonal regeneration.
PTEN resulted in neurites regenerating over a long distance, extending from the lesion site towards the optic chiasm. Fluorescence images of longitudinal sections of the optic nerve showing axonal regeneration 6 weeks after optic nerve axotomy in rats intravitreally injected with vector 4 weeks before lesioning. D Quantification of the fluorescence intensity at different distances to the lesion site.
Click here to view Figure 7: CTB-FITC labeling for regenerating axons in the brain 6 weeks after optic nerve axotomy in rats intravitreally injected with vector 4 weeks before lesioning. Therefore, we evaluated the expression of pS6 by western blot assay and immunohistochemistry. At 4 weeks after injection, analysis of the percentage of pS6-positive cells in the ganglion cell layer revealed significant differences among Wt AAV2-GFP 7.
Western blot assay for retinal expression of pS6 ribosomal protein and GLAST 6 weeks after optic nerve axotomy in rats intravitreally injected with vector 4 weeks before lesioning. Click here to view Figure 9: Immunolabeling for pS6 ribosomal protein in retinas 4 weeks after intravitreal injection of AAV2 vectors.
PTEN immediately after optic nerve axotomy. Six weeks later, the density of RGCs was quantified and regenerating axons were examined. PTEN compared with pre-axotomy injection of the vector. PTEN resulted in less axonal regeneration at every analyzed region of the optic nerve compared with pre-axotomy injection.
It has been shown that AAV2 strongly depends on heparan sulfate proteoglycans for transduction Zaiss et al. Once in the retina, AAV2 needs to overcome additional barriers to achieve efficient transduction.
The ubiquitin-proteasome pathway is a major obstacle to AAV-mediated gene expression. This pathway degrades the viral particles during their intracellular trafficking from the cytoplasm to the nucleus, and involves the phosphorylation of tyrosine residues by the epidermal growth factor receptor Ding et al. Thus, substitution of certain surface-exposed tyrosine residues on AAV2 capsids may allow the vectors to escape ubiquitination and proteasomal degradation.
Interestingly, mutation of surface-exposed tyrosine residues on the AAV2 capsid can also lead to more efficient transduction of RGCs Zhong et al. AAV2 is the only serotype able to transduce RGCs efficiently, making it the only effective vector for therapies targeting glaucomatous optic neuropathy Harvey et al.
RGC apoptosis is the common result of glaucomatous optic neuropathy and traumatic optic neuropathy, although the underlying mechanisms may be different. Moreover, the progression of glaucomatous optic neuropathy is slow, while that of traumatic optic neuropathy is relatively rapid.
The more potent transduction efficiency of YF AAV2 makes it a suitable gene delivery vector for treating traumatic optic neuropathy. However, conditional gene deletion prior to optic nerve injury is impossible to translate to clinical practice, while therapies based on RNA interference to knockdown a target gene may be most useful for treatment of optic nerve damage Guzman-Aranguez et al. Previous and more recent studies have shown that optic nerve crush leads to an increase in retinal extracellular glutamate to neurotoxic levels Vorwerk et al.
The mechanisms underlying this protective effect are unclear. PTEN-injected rats, with some regenerating axons regrowing towards the optic tract. PTEN were less significant compared with pre-axotomy injection. It also induced axons to regenerate up to near the optic chiasm, indicating its potential for clinical treatment. In summary, our findings show that YF AAV2 is a promising vector for gene therapy for traumatic optic neuropathy. YF AAV2-mediated PTEN knockdown was able to activate mTOR complex-1 and induce long-distance axonal regeneration in wildtype animals, suggesting that it is a promising translatable treatment for traumatic optic neuropathy.
In view of the complexity of the cell and molecular mechanisms underlying axonal regeneration, future studies will focus on using YF AAV2 to modulate mTOR complex-1 as well as other critical targets to achieve robust axonal regeneration for functional visual recovery.