Shreyasi Choudhury

ER Stress Is Involved in T17M Rhodopsin-Induced Retinal Degeneration

PURPOSE The human rhodopsin (Rho) mutation T17M leads to autosomal dominant retinitis pigmentosa (adRP). The goal of our study was to elucidate the role of endoplasmic reticulum (ER) stress in retinal degeneration in hT17M Rho mice and identify potential candidates for adRP gene therapy. METHODS We used transgenic mice expressing the ER stress-activated indicator (ERAI) and hT17M Rho to evaluate the activation of ER stress responses. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze changes in the expression of 30 unfolded protein response (UPR)-associated genes at P12, 15, 18, 21, and 25. The cytosolic fraction of hT17M Rho retinal cells was used to measure the release of cytochrome C and apoptotic inducing factor-1 (AIF1) by Western blotting. Optical coherence tomography (OCT) analysis was performed for 1-month-old hT17M Rho mice. RESULTS hT17M Rho was localized in the outer nuclear layer (ONL) of T17M(+/-)ERAI(+/-) photoreceptors as well as C57BL/6 retinas injected with AAV-hT17M Rho-GFP. In P15 hT17M Rho retinas, we observed an up-regulation of UPR genes (Atf4, Eif2α, Xbp1, Bip, Canx, and Hsp90), autophagy genes and proapoptotic Bcl2 genes. OCT, and the downregulation of Nrl and Crx gene expression confirmed that cell death occurs in 55% of photoreceptors via the up-regulation of caspase-3 and caspase-12, and the release of AIF1 from the mitochondria. CONCLUSIONS The ER stress response is involved in retinal degeneration in hT17M Rho mice. The final demise of photoreceptors occurs via apoptosis involving ER stress-associated and mitochondria-induced caspase activation. We identified Atg5, Atg7, Bax, Bid, Bik, and Noxa as potential therapeutic targets for adRP treatment.

Caspase-7 ablation modulates UPR, reprograms TRAF2-JNK apoptosis and protects T17M rhodopsin mice from severe retinal degeneration

The UPR is activated in the mouse retina expressing misfolded T17M rhodopsin (RHO) during autosomal dominant retinitis pigmentosa (ADRP) progression. Therefore, the goal of this study is to validate the UPR-induced caspase-7 as a new therapeutic target that modulates the UPR, reduces the level of apoptosis and protects the ADRP retina from retinal degeneration and light-induced damage. Mice were analyzed using ERG, SD-OCT and histology to determine the role of caspase-7 ablation. The results of these experiments demonstrate the significant preservation of photoreceptors and their function in T17M RHO CASP-7 retinas from P30 to P90 compared with control mice. These mice were also protected from the light-induced decline in the ERG responses and apoptosis. The RNA and protein analyses of T17M RHO+Csp7-siRNA, Tn+Csp7-siRNA 661W cells and T17M RHO CASP-7 retinas revealed that caspase-7 ablation reprograms the UPR and reduces JNK-induced apoptosis. This reduction is believed to occur through the downregulation of the mTOR and Hif1a proteins. In addition, decline in activated PARP1 was detected in T17M RHO CASP-7 retina. Altogether, our findings indicate that the targeting of caspase-7 in T17M RHO mice could be a feasible therapeutic strategy for advanced stages of ADRP.

Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors.

ABSTRACT Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection. IMPORTANCE AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors that can mediate transduction following noninvasive, intravitreal injection. HS binding has been postulated to play a role in intravitreally mediated transduction of retina. Our evaluation of the HS binding of AAV2-based variants and other AAV serotype vectors and the correlation of this property with transduction points to HS affinity as a factor controlling retinal transduction following Ivt delivery. However, HS binding is not the only requirement for improved Ivt-mediated transduction. We show that AAV2-based vectors lacking heparin binding transduce retina by subretinal injection and display a remarkable ability to transduce photoreceptors, indicating that other receptors are involved in this phenotype.

Targeting Caspase-12 to Preserve Vision in Mice With Inherited Retinal Degeneration

PURPOSE The unfolded protein response is known to contribute to the inherited retinal pathology observed in T17M rhodopsin (T17M) mice. Recently it has been demonstrated that the endoplasmic reticulum stress-associated caspase-12 is activated during progression of retinal degeneration in different animal models. Therefore, we wanted to explore the role of caspase-12 in the mechanism of retinopathy in T17M mice and determine if inhibiting apoptosis in this way is a viable approach for halting retinal degeneration. METHODS One, two-, and three-month-old C57BL6/J, caspase-12-/-, T17M, and T17M caspase-12-/- mice were analyzed by scotopic ERG, spectral-domain optical coherence tomography (SD-OCT), histology, quantitative (q)RT-PCR, and Western blot of retinal RNA and protein extracts. Calpain and caspase-3/7 activity assays were measured in postnatal (P) day 30 retinal extracts. RESULTS Caspase-12 ablation significantly prevented a decline in the a- and b-wave ERG amplitudes in T17M mice during three months, increasing the amplitudes from 232% to 212% and from 160% to 138%, respectively, as compared to T17M retinas. The SD-OCT results and photoreceptor row counts demonstrated preservation of retinal structural integrity and postponed photoreceptor cell death. The delay in photoreceptor cell death was due to significant decreases in the activity of caspase-3/7 and calpain, which correlated with an increase in calpastatin expression. CONCLUSIONS We validated caspase-12 as a therapeutic target, ablation of which significantly protects T17M photoreceptors from deterioration. Although the inhibition of apoptotic activity alone was not sufficient to rescue T17M photoreceptors, in combination with other nonapoptotic targets, caspase-12 could be used to treat inherited retinopathy.

Modulation of the Rate of Retinal Degeneration in T17M RHO Mice by Reprogramming the Unfolded Protein Response.

The goal of this study is to validate whether reprogramming of the UPR via modulation of pro-apoptotic caspase-7 and CHOP proteins could be an effective approach to slow down the rate of retinal degeneration in ADRP mice. In order to pursue our goal we created the T17M RHO CASP7 and T17M RHO CHOP mice to study the impact of the CASP7 or CHOP ablations in T17M RHO retina by ERG, SD-OCT, histology and western blot analysis. The scotopic ERG demonstrated that the ablation of the CASP7 in T17M RHO retina leads to significant preservation of the function of photoreceptors compared to control. Surprisingly, the ablation of pro-apoptotic CHOP protein in T17M RHO mice led to a more severe form of retinal degeneration. Results of the SD-OCT and histology were in agreement with the ERG data. The further analysis demonstrated that the preservation of the structure and function or the acceleration of the onset of the T17M RHO photoreceptor degeneration occurred via reprogramming of the UPR. In addition, the CASP7 ablation leads to the inhibition of cJUN mediated apoptosis, while the ablation of CHOP induces an increase in the HDAC. Thus, manipulation with the UPR requires careful examination in order to achieve a therapeutic effect.

Novel Methodology for Creating Macaque Retinas with Sortable Photoreceptors and Ganglion Cells

Purpose: The ability to generate macaque retinas with sortable cell populations would be of great benefit to both basic and translational studies of the primate retina. The purpose of our study was therefore to develop methods to achieve this goal by selectively labeling, in life, photoreceptors (PRs) and retinal ganglion cells (RGCs) with separate fluorescent markers. Methods: Labeling of macaque (Macaca fascicularis) PRs and RGCs was accomplished by subretinal delivery of AAV5-hGRK1-GFP, and retrograde transport of micro-ruby™ from the lateral geniculate nucleus, respectively. Retinas were anatomically separated into different regions. Dissociation conditions were optimized, and cells from each region underwent fluorescent activated cell sorting (FACS). Expression of retinal cell type- specific genes was assessed by quantitative real-time PCR to characterize isolated cell populations. Results: We show that macaque PRs and RGCs can be simultaneously labeled in-life and enriched populations isolated by FACS. Recovery from different retinal regions indicated efficient isolation/enrichment for PRs and RGCs, with the macula being particularly amendable to this technique. Conclusions: The methods and materials presented here allow for the identification of novel reagents designed to target RGCs and/or photoreceptors in a species that is phylogenetically and anatomically similar to human. These techniques will enable screening of intravitreally-delivered AAV capsid libraries for variants with increased tropism for PRs and/or RGCs and the evaluation of vector tropism and/or cellular promoter activity of gene therapy vectors in a clinically relevant species.