Marina S. Gorbatyuk

Restoration of visual function in P23H rhodopsin transgenic rats by gene delivery of BiP/Grp78

The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2α and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2α, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

In Vivo RNAi-Mediated α-Synuclein Silencing Induces Nigrostriatal Degeneration

Two small-interfering RNAs (siRNAs) targeting α-synuclein (α-syn) and three control siRNAs were cloned in an adeno-associated virus (AAV) vector and unilaterally injected into rat substantia nigra pars compacta (SNc). Reduction of α-syn resulted in a rapid (4 week) reduction in the number of tyrosine hydroxylase (TH) positive cells and striatal dopamine (DA) on the injected side. The level of neurodegeneration induced by the different siRNAs correlated with their ability to downregulate α-syn protein and mRNA in tissue culture and in vivo. Examination of various SNc neuronal markers indicated that neurodegeneration was due to cell loss and not just downregulation of DA synthesis. Reduction of α-syn also resulted in a pronounced amphetamine induced behavioral asymmetry consistent with the level of neurodegeneration. In contrast, none of the three control siRNAs, which targeted genes not normally expressed in SNc, showed evidence of neurodegeneration or behavioral asymmetry, even at longer survival times. Moreover, co-expression of both rat α-syn and α-syn siRNA partially reversed the neurodegenerative and behavioral effects of α-syn siRNA alone. Our data show that α-syn plays an important role in the rat SNc and suggest that both up- and downregulation of wild-type α-syn expression increase the risk of nigrostriatal pathology.Two small-interfering RNAs (siRNAs) targeting alpha-synuclein (alpha-syn) and three control siRNAs were cloned in an adeno-associated virus (AAV) vector and unilaterally injected into rat substantia nigra pars compacta (SNc). Reduction of alpha-syn resulted in a rapid (4 week) reduction in the number of tyrosine hydroxylase (TH) positive cells and striatal dopamine (DA) on the injected side. The level of neurodegeneration induced by the different siRNAs correlated with their ability to downregulate alpha-syn protein and mRNA in tissue culture and in vivo. Examination of various SNc neuronal markers indicated that neurodegeneration was due to cell loss and not just downregulation of DA synthesis. Reduction of alpha-syn also resulted in a pronounced amphetamine induced behavioral asymmetry consistent with the level of neurodegeneration. In contrast, none of the three control siRNAs, which targeted genes not normally expressed in SNc, showed evidence of neurodegeneration or behavioral asymmetry, even at longer survival times. Moreover, co-expression of both rat alpha-syn and alpha-syn siRNA partially reversed the neurodegenerative and behavioral effects of alpha-syn siRNA alone. Our data show that alpha-syn plays an important role in the rat SNc and suggest that both up- and downregulation of wild-type alpha-syn expression increase the risk of nigrostriatal pathology.

Glucose regulated protein 78 diminishes α-synuclein neurotoxicity in a rat model of Parkinson disease.

Accumulation of human wild-type (wt) α-synuclein (α-syn) induces neurodegeneration in humans and in experimental rodent models of Parkinson disease (PD). It also leads to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). We overexpressed glucose regulated protein 78, also known as BiP (GRP78/BiP), to test the hypothesis that this ER chaperone modulates the UPR, blocks apoptosis, and promotes the survival of nigral dopamine (DA) neurons in a rat model of PD induced by elevated level of human α-syn. We determined that α-syn activates ER stress mediators associated with pancreatic ER kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) signaling pathways as well as proaoptotic CCAAT/-enhancer-binding protein homologous protein (CHOP) in nigral DA neurons. At the same time, overexpression of GRP78/BiP diminished α-syn neurotoxicity by down regulating ER stress mediators and the level of apoptosis, promoted survival of nigral tyrosine hydroxylase (TH) positive cells and resulted in higher levels of striatal DA, while eliminating amphetamine induced behavioral asymmetry. We also detected a complex between GRP78/BiP and α-syn that may contribute to prevention of the neurotoxicity caused by α-syn. Our data suggest that the molecular chaperone GRP78/BiP plays a neuroprotective role in α-syn-induced Parkinson-like neurodegeneration.

AAV Delivery of Wild-Type Rhodopsin Preserves Retinal Function in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa

Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / +  or RHO + /- backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.

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.

Suppression of mouse rhodopsin expression in vivo by AAV mediated siRNA delivery.

PURPOSE The purpose of this study is to demonstrate that the expression of rhodopsin can be down regulated in vivo by AAV-delivered siRNA. This is the first step in an RNA replacement strategy for the allele-independent treatment of Autosomal Dominant Retinitis Pigmentosa (ADRP). METHODS HEK 293 cells were co-transfected with a plasmid carrying mouse RHO cDNA driven by the CMV promoter and a chemically synthesized siRNA duplex of 21 nucleotides. Reduction of RHO mRNA was confirmed by RT-PCR. One active siRNA and a control siRNA were embedded in a small hairpin RNA (shRNA) and cloned in Adeno-associated virus (AAV) vector under regulation of the H1 promoter and containing a GFP reporter. AAV5 expressing either active siRNA or an irrelevant siRNA were subretinaly injected into the right eyes of wild-type or RHO+/- heterozygote mice at post-natal day 16. At 1 and 2 months post-injection, animals were analyzed by electroretinography (ERG). Animals were then sacrificed, and retinas were examined by Western blot, RT-PCR, histology and immunohistochemistry. RESULTS All of the siRNAs tested in HEK 293 cells caused degradation of RHO mRNA, although the efficiency varied from 25% to 80%. In vivo siRNA delivery to the retina led to more than 40% reduction of scotopic a- and b-wave amplitudes in RHO+/- heterozygotes. Although the reduction of RHO mRNA was estimated at 30% compared to control animals, Western blots revealed 60% decrease in rhodopsin content. Histological analysis showed significant reduction in the thickness of the ONL, ranging between 53% and 86%. CONCLUSIONS AAV-siRNA delivery into the subretinal space resulted in the reduction of retinal function caused by diminished RHO mRNA and protein content. This level of reduction may permit the replacement of endogenous mRNA with siRNA-resistant mRNA encoding wild-type RHO.

The Molecular Chaperone GRP78/BiP as a Therapeutic Target for Neurodegenerative Disorders: A Mini Review

The glucose regulated protein 78 (GRP78), also known as BiP, is the endoplasmatic reticulum (ER) homologue of HSP70, which plays a dual role in the ER by controlling protein folding, in order to prevent aggregation, and by regulating the signaling of the unfolded protein response (UPR). Most neurodegenerative disorders including Parkinsons, Alzheimers diseases and progressive retinal degeneration are characterized by activation of the UPR and modified expression of GRP78. The expression levels and activity of GRP78 are altered with age raising the question of whether the lack of GRP78 could be a predisposing factor for many neurodegenerative disorders associated with age including PD, Alzheimer and Age-related macular degeneration. Attempts to induce or upregulate GRP78 in animal models of neurodegeneration have recently been made with the help of pharmacological BiP protein Inducer X (BIX) and GRP78 cDNA delivery via adeno-associated virus (AAV) vectors. The results of these studies validate GRP78 as a new therapeutic target for treatments of forebrain ischemia, Parkinson disease and retinal degeneration. These data, together with the results from age-related studies, highlight the importance for developing drugs to induce elevation of endogenous GRP78 in order to increase cellular survival and extend functional longevity.

ER Stress in Retinal Degeneration in S334ter Rho Rats

The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12–P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.

Long-Term Rescue of Retinal Structure and Function by Rhodopsin RNA Replacement with a Single Adeno-Associated Viral Vector in P23H RHO Transgenic Mice

Many mutations in the human rhodopsin gene (RHO) cause autosomal dominant retinitis pigmentosa (ADRP). Our previous studies with a P23H (proline-23 substituted by histidine) RHO transgenic mouse model of ADRP demonstrated significant improvement of retinal function and preservation of retinal structure after transfer of wild-type rhodopsin by AAV. In this study we demonstrate long-term rescue of retinal structure and function by a single virus expressing both RHO replacement cDNA and small interfering RNA (siRNA) to digest mouse Rho and human P23H RHO mRNA. This combination should prevent overexpression of rhodopsin, which can be deleterious to photoreceptors. On the basis of the electroretinogram (ERG) response, degeneration of retinal function was arrested at 2 months postinjection, and the response was maintained at this level until termination at 9 months. Preservation of the ERG response in P23H RHO mice reflected survival of photoreceptors: both the outer nuclear layer (ONL) and outer segments of photoreceptor cells maintained the same thickness as in nontransgenic mice, whereas the control injected P23H eyes exhibited severe thinning of the ONL and outer segments. These findings suggest that delivery of both a modified cDNA and an siRNA by a single adeno-associated viral vector provided long-term rescue of ADRP in this model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease.

Induction of Endoplasmic Reticulum Stress Genes, BiP and Chop, in Genetic and Environmental Models of Retinal Degeneration

PURPOSE Endoplasmic reticulum (ER) stress has been observed in animal models of retinitis pigmentosa expressing P23H rhodopsin. We compared levels of tightly induced ER stress genes, Binding of immunoglobulin protein (BiP) and CCAAT/enhancer-binding protein homologous protein (Chop), in seven additional models of retinal degeneration arising from genetic or environmental causes. METHODS Retinas from transgenic S334ter rhodopsin (lines 3, 4, and 5) and Royal College of Surgeons (RCS and RCS-p+) rats from postnatal (P) days 10 to 120 were analyzed. In a constant light (CL) model of retinal degeneration, BALB/c mice were exposed to 15,000 lux of CL for 0 to 8 hours. Retinal tissues from three to eight animals per experimental condition were collected for histologic and molecular analyses. RESULTS S334ter animals revealed significant increases in BiP, S334ter-3 (3.3× at P15), S334ter-4 (4× at P60), and S334ter-5 (2.2× at P90), and Chop, S334ter-3 (1.3× at P15), S334ter-4 (1.5× at P30), and S334ter-5 (no change), compared with controls. P23H-3 rats showed significant increase of BiP at P60 (2.3×) and Chop (1.6×). RCS and RCS-p+ rats showed significant increases in BiP at P60 (2.4×) and P20 (1.8×), respectively, but no statistically significant changes in Chop. BALB/c mice showed increases in BiP (1.5×) and Chop (1.3×) after 4 hours of CL. Increased levels of these ER stress markers correlated with photoreceptor cell loss. CONCLUSIONS Our study reveals surprising increases in BiP and to a lesser degree Chop in retinal degenerations arising from diverse causes. We propose that manipulation of ER stress responses may be helpful in treating many environmental and heritable forms of retinal degeneration.