Retinitis Pigmentosa is a common form of hereditary retinal degeneration constituting the largest Mendelian genetic cause of blindness in the developed world. It has been widely suggested that oxidative stress possibly contributes to its pathogenesis. We measured the levels of total antioxidant capacity, free nitrotyrosine, thiobarbituric acid reactive substances (TBARS) formation, extracellular superoxide dismutase (SOD3) activity, protein, metabolites of the nitric oxide/cyclic GMP pathway, heme oxygenase-I and inducible nitric oxide synthase expression in aqueous humor or/and peripheral blood from fifty-six patients with retinitis pigmentosa and sixty subjects without systemic or ocular oxidative stress-related disease. Multivariate analysis of covariance revealed that retinitis pigmentosa alters ocular antioxidant defence machinery and the redox status in blood. Patients with retinitis pigmentosa present low total antioxidant capacity including reduced SOD3 activity and protein concentration in aqueous humor. Patients also show reduced SOD3 activity, increased TBARS formation and upregulation of the nitric oxide/cyclic GMP pathway in peripheral blood. Together these findings confirmed the hypothesis that patients with retinitis pigmentosa present reduced ocular antioxidant status. Moreover, these patients show changes in some oxidative-nitrosative markers in the peripheral blood. Further studies are needed to clarify the relationship between these peripheral markers and retinitis pigmentosa.
Oxidative stress is one of the major factors that trigger photoreceptor apoptosis. To investigate whether resveratrol, a potent antioxidant and small molecule activator of the FoxO pathway, would be neuroprotective against photoreceptor cell death in a rodent model of retinal detachment.
Retinal detachment was created in adult Brown Norway rats by subretinal injection of sodium hyaluronate. The animals were treated daily with vehicle or resveratrol (20mg/kg) intraperitoneal injection. Photoreceptor death was assessed by counting the number of apoptotic cells with TdT-dUTP terminal nick-end labeling (TUNEL) and measurement of the outer nuclear layer (ONL) thickness 3 days after RD. Changes in expression of FoxO1a, FoxO3a, and FoxO4 were analyzed by western blot. The activity of caspase 3, caspase 8, caspase 9, spectrin and their cleavage forms were studied.
Three days after retinal detachment, caspase 3, caspase 8 and caspase 9 were significantly activated in the detached retina. Spectrin cleavage products at 120 and 145 kDa were also detected. Both caspase and calpain activation are involved in apoptotic photoreceptor cell death in detached retinas. Treatment with resveratrol increases FoxO1a, FoxO3a, and FoxO4 protein expression in detached retinas only. Resveratrol treatment decreases activation of intrinsic and extrinsic caspase apoptotic pathways triggered by RD. The number of TUNEL-positive cells decreases from 1301±51 cells/mm2 in control groups to 430±35 cells/mm2 in treatment groups (p<0.05). Resveratrol treatment also demonstrates 59% less ONL thickness loss compared to controls.
Resveratrol treatment up-regulates the FoxO family and blocks Caspase3, 8, and 9 activation. Resveratrol has the potential to be used as a novel therapeutic agent for preventing vision loss in diseases characterized by photoreceptor detachment.
Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice.
I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated.
Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration.
Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease.
Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.
In eukaryotes the genetic material is enclosed by a continuous membrane system, the nuclear envelope (NE). Along the NE specific proteins assemble to form meshworks and mutations in these proteins have been described in a group of human diseases called laminopathies. Laminopathies include lipodystrophies, muscle and cardiac diseases as well as metabolic or progeroid syndromes. Most laminopathies are caused by mutations in the LMNAgene encoding lamins A/C. Together with Nesprins (Nuclear Envelope Spectrin Repeat Proteins) they are core components of the LINC complex (Linker of Nucleoskeleton and Cytoskeleton). The LINC complex connects the nucleoskeleton and the cytoskeleton and plays a role in the transfer of mechanically induced signals along the NE into the nucleus, and its components have been attributed functions in maintaining nuclear and cellular organization as well as signal transduction.
Here we narrowed down the interaction sites between lamin A and Nesprin-2 to aa 403–425 in lamin A and aa 6146–6347 in Nesprin-2. Laminopathic mutations in and around the involved region of lamin A (R401C, G411D, G413C, V415I, R419C, L421P, R427G, Q432X) modulate the interaction with Nesprin-2 and this may contribute to the disease phenotype. The most notable mutation is the lamin A mutation Q432X that alters LINC complex protein assemblies and causes chromosomal and transcription factor rearrangements.
Mutations in Nesprin-2 and lamin A are characterised by complex genotype phenotype relations. Our data show that each mutation in LMNAanalysed here has a distinct impact on the interaction among both proteins that substantially explains how distinct mutations in widely expressed genes lead to the formation of phenotypically different diseases.
To study bilateral nerve changes in a newly developed novel mouse model for neurotrophic keratopathy by approaching the trigeminal nerve from the lateral fornix. Surgical axotomy of the ciliary nerve of the trigeminal nerve was performed in adult BALB/c mice at the posterior sclera. Axotomized, contralateral, and sham-treated corneas were excised on post-operative days 1, 3, 5, 7 and 14 and immunofluorescence histochemistry was performed with anti-β-tubulin antibody to evaluate corneal nerve density. Blink reflex was evaluated using a nylon thread. The survival rate was 100% with minimal bleeding during axotomy and a surgical time of 8±0.5 minutes. The blink reflex was diminished at day 1 after axotomy, but remained intact in the contralateral eyes in all mice. The central and peripheral subbasal nerves were not detectable in the axotomized cornea at day 1 (p<0.001), compared to normal eyes (101.3±14.8 and 69.7±12.0 mm/mm2 centrally and peripherally). Interestingly, the subbasal nerve density in the contralateral non-surgical eyes also decreased significantly to 62.4±2.8 mm/mm2 in the center from day 1 (p<0.001), but did not change in the periphery (77.3±11.7 mm/mm2, P = 0.819). Our novel trigeminal axotomy mouse model is highly effective, less invasive, rapid, and has a high survival rate, demonstrating immediate loss of subbasal nerves in axotomized eyes and decreased subbasal nerves in contralateral eyes after unilateral axotomy. This model will allow investigating the effects of corneal nerve damage and serves as a new model for neurotrophic keratopathy.
Oxidative stress in the retinal pigment epithelium (RPE) is hypothesized to be a major contributor to the development of age-related macular degeneration (AMD). Mitochondrial manganese superoxide dismutase (MnSOD) is a critical antioxidant protein that scavenges the highly reactive superoxide radical. We speculated that specific reduction of MnSOD in the RPE will increase the level of reactive oxygen species in the retina/RPE/choroid complex leading to pathogenesis similar to geographic atrophy. To test this hypothesis, an Sod2-specific hammerhead ribozyme (Rz), delivered by AAV2/1 and driven by the human VMD2 promoter was injected subretinally into C57BL/6J mice. Dark-adapted full field electroretinogram (ERG) detected a decrease in the response to light. We investigated the age-dependent phenotypic and morphological changes of the outer retina digital fundus imaging and SD-OCT measurement of ONL thickness. Fundus microscopy revealed pigmentary abnormalities in the retina and these corresponded to sub-retinal and sub-RPE deposits seen in SD-OCT B-scans. Light and electron microscopy documented the localization of apical deposits and thickening of the RPE. In RPE flat-mounts we observed abnormally displaced nuclei and regions of apparent fibrosis in the central retina of the oldest mice. This region was surrounded by enlarged and irregular RPE cells that have been observed in eyes donated by AMD patients and in other mouse models of AMD.
retinal pigment epithelium; oxidative stress; superoxide dismutase; mitochondria; mouse model; age related macular degeneration
Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea.
The rapid and massive degeneration of photoreceptors in retinal degeneration might have a dramatic negative effect on retinal circuits downstream of photoreceptors. However, the impact of photoreceptor loss on the morphology and function of retinal ganglion cells (RGCs) is not fully understood, precluding the rational design of therapeutic interventions that can reverse the progressive loss of retinal function. The present study investigated the morphological changes in several identified RGCs in the retinal degeneration rd1 mouse model of retinitis pigmentosa (RP), using a combination of viral transfection, microinjection of neurobiotin and confocal microscopy. Individual RGCs were visualized with a high degree of detail using an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP), allowed for large-scale surveys of the morphology of RGCs over a wide age range. Interestingly, we found that the RGCs of nine different types we encountered were especially resistant to photoreceptor degeneration, and retained their fine dendritic geometry well beyond the complete death of photoreceptors. In addition, the RGC-specific markers revealed a remarkable degree of stability in both morphology and numbers of two identified types of RGCs for up to 18 months of age. Collectively, our data suggest that ganglion cells, the only output cells of the retina, are well preserved morphologically, indicating the ganglion cell population might be an attractive target for treating vision loss.
Dry eye disease can cause ocular surface inflammation that disrupts the corneal epithelial barrier. While dry eye patients are known to have an increased risk of corneal infection, it is not known whether there is a direct causal relationship between these two conditions. Here, we tested the hypothesis that experimentally-induced dry eye (EDE) increases susceptibility to corneal infection using a mouse model. In doing so, we also examined the role of surfactant protein D (SP-D), which we have previously shown is involved in corneal defense against infection. Scopolamine injections and fan-driven air were used to cause EDE in C57BL/6 or Black Swiss mice (wild-type and SP-D gene-knockout). Controls received PBS injections and were housed normally. After 5 or 10 days, otherwise uninjured corneas were inoculated with 109 cfu of Pseudomonas aeruginosa strain PAO1. Anesthesia was maintained for 3 h post-inoculation. Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation. SP-D was measured by Western immunoblot, and corneal pathology assessed from 6 h to 4 days. EDE mice showed reduced tear volumes after 5 and 10 days (each by ∼75%, p<0.001) and showed fluorescein staining (i.e. epithelial disruption). Surprisingly, there was no significant difference in corneal pathology between EDE mice and controls (∼10–14% incidence). Before bacterial inoculation, EDE mice showed elevated SP-D in ocular washes. After inoculation, fewer bacteria were recovered from ocular washes of EDE mice (<2% of controls, p = 0.0004). Furthermore, SP-D knockout mice showed a significant increase in P. aeruginosa corneal colonization under EDE conditions. Taken together, these data suggest that SP-D contributes to corneal defense against P. aeruginosa colonization and infection in EDE despite the loss of barrier function to fluorescein.
Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-β and mediates most key fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues and wounded corneas.
Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5′ Rapid Amplification of cDNA Ends (RACE).
HCF stimulated by TGF-β contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle “hinge” region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11.
Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.
Proteolytic processing of connective tissue growth factor (CTGF, 38 kDa) alters its biological effects. C-terminal fragments of CTGF (21 kDa and 25 kDa) are abundant in normal ocular tissues, and peak 11 days after excimer laser ablation in rat corneas. Processing of CTGF during corneal wound healing may impact scar formation.
Excessive scarring (fibrosis) is a major cause of pathologies in multiple tissues, including lung, liver, kidney, heart, cornea, and skin. The transforming growth factor- β (TGF- β) system has been shown to play a key role in regulating the formation of scar tissue throughout the body. Furthermore, connective tissue growth factor (CTGF) has been shown to mediate most of the fibrotic actions of TGF- β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. Currently, no approved drugs selectively and specifically regulate scar formation. Thus, there is a need for a drug that selectively targets the TGF- β cascade at the molecular level and has minimal off-target side effects. This chapter focuses on the design of hammerhead ribozymes, measurement of kinetic activity, and assessment of knockdown mRNAs of TGF- β and CTGF in cell cultures.
Ribozymes; TGF- β; CTGF; Scar formation; Transduction; Oligonucleotides
The ANP32 family of proteins have been implicated in neuronal function through biochemical and cellular biology studies in neurons, as well as by recent behavioural studies of a gene-trapped loss-of-function mutation of Anp32e in mice, particularly with respect to fine motor function. A second targeted allele of the Anp32e, however, did not appear to demonstrate neurological phenotypes.
Using a stringently controlled cohort of ten-generation backcrossed, co-caged, sex-matched, littermate pairs, we assayed for potential motor defects in the targeted ANP32E-deficient mice. We found no phenotypic difference in any assays.
Since it is unlikely that the gene-trap is a more complete loss-of-function, our results suggest that ANP32E has no appreciable effect on motor functions and that genetic background differences most likely account for the gene-trap phenomena.
The activation of caspase-3 is an important hallmark in Parkinson’s disease. It could induce neuron death by apoptosis and microglia activation by inflammation. As a result, inhibition the activation of caspase-3 would exert synergistic dual effect in brain in order to prevent the progress of Parkinson’s disease. Silencing caspase-3 genes by RNA interference could inhibit the activation of caspase-3. We developed a brain-targeted gene delivery system based on non-viral gene vector, dendrigraft poly-L-lysines. A rabies virus glycoprotein peptide with 29 amino-acid linked to dendrigraft poly-L-lysines could render gene vectors the ability to get across the blood brain barrier by specific receptor mediated transcytosis. The resultant brain-targeted vector was complexed with caspase-3 short hairpin RNA coding plasmid DNA, yielding nanoparticles. In vivo imaging analysis indicated the targeted nanoparticles could accumulate in brain more efficiently than non-targeted ones. A multiple dosing regimen by weekly intravenous administration of the nanoparticles could reduce activated casapse-3 levels, significantly improve locomotor activity and rescue dopaminergic neuronal loss and in Parkinson’s disease rats’ brain. These results indicated the rabies virus glycoprotein peptide modified brain-targeted nanoparticles were promising gene delivery system for RNA interference to achieve anti-apoptotic and anti-inflammation synergistic therapeutic effects by down-regulation the expression and activation of caspase-3.
Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs), a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1) can prevent loss of RGCs in the rat retina with optic nerve transection (ONT) and in cultures of RGC-5 cells with CoCl2 injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl2. The expression of HIF-1α that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.
Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1st or 2nd decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.
Pharmacologic inhibition of aldose reductase (AR) previously has been studied with respect to diabetic retinopathy with mixed results. Since drugs can have off-target effects, we studied the effects of AR deletion on the development and molecular abnormalities that contribute to diabetic retinopathy. Since recent data suggests an important role for leukocytes in the development of the retinopathy, we determined also if AR in leukocytes contributes to leukocyte-mediated death of retinal endothelial cells in diabetes.
Wild-type (WT; C57BL/6J) and AR deficient (AR−/−) mice were made diabetic with streptozotocin. Mice were sacrificed at 2 and 10 months of diabetes to evaluate retinal vascular histopathology, to quantify retinal superoxide production and biochemical and physiological abnormalities in the retina, and to assess the number of retinal endothelial cells killed by blood leukocytes in a co-culture system.
Diabetes in WT mice developed the expected degeneration of retinal capillaries, and increased generation of superoxide by the retina. Leukocytes from diabetic WT mice also killed more retinal endothelial cells than did leukocytes from nondiabetic animals (p<0.0001). Deletion of AR largely (P<0.05) inhibited the diabetes-induced degeneration of retinal capillaries, as well as the increase in superoxide production by retina. AR-deficiency significantly inhibited the diabetes-induced increase in expression of inducible nitric oxide synthase (iNOS) in retina, but had no significant effect on expression of intercellular adhesion molecule-1 (ICAM-1), phosphorylated p38 MAPK, or killing of retinal endothelial cells by leukocytes.
AR contributes to the degeneration of retinal capillaries in diabetic mice. Deletion of the enzyme inhibits the diabetes-induced increase in expression of iNOS and of superoxide production, but does not correct a variety of other pro-inflammatory abnormalities associated with the development of diabetic retinopathy.
Widespread gene delivery to the retina is an important challenge for the treatment of retinal diseases, such as retinal dystrophies. We and others have recently shown that the intravenous injection of a self-complementary (sc) AAV9 vector can direct efficient cell transduction in the central nervous system, in both neonatal and adult animals. We show here that the intravenous injection of scAAV9 encoding green fluorescent protein (GFP) resulted in gene transfer to all layers of the retina in adult mice, despite the presence of a mature blood-eye barrier. Cell morphology studies and double-labeling with retinal cell-specific markers showed that GFP was expressed in retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells and retinal ganglion cells. The cells on the inner side of the retina, including retinal ganglion cells in particular, were transduced with the highest efficiency. Quantification of the cell population co-expressing GFP and Brn-3a showed that 45% of the retinal ganglion cells were efficiently transduced after intravenous scAAV9-GFP injection in adult mice. This study provides the first demonstration that a single intravenous scAAV9 injection can deliver transgenes to the retinas of both eyes in adult mice, suggesting that this vector serotype is able to cross mature blood-eye barriers. This intravascular gene transfer approach, by eliminating the potential invasiveness of ocular surgery, could constitute an alternative when fragility of the retina precludes subretinal or intravitreal injections of viral vectors, opening up new possibilities for gene therapy for retinal diseases.
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.
Mao and colleagues show that a single subretinal injection of AAV serotype 5 (AAV5) vector encoding both a small interfering RNA targeted against a mutant form of rhodopsin (P23H RHO) and a replacement cDNA for the nonmutant form of RHO preserves retinal structure and leads to full recovery of retinal function in a mouse model of autosomal dominant retinitis pigmentosa.
The accumulation of β-amyloid peptides in the brain has been recognized as an essential factor in Alzheimer’s disease pathology. Several proteases, including Neprilysin (NEP), endothelin converting enzyme (ECE), and insulin degrading enzyme (IDE), have been shown to cleave β-amyloid peptides (Aβ). We have previously reported reductions in amyloid in APP+PS1 mice with increased expression of ECE. In this study we compared the vector-induced increased expression of NEP and IDE. We used recombinant adeno-associated viral vectors expressing either native forms of NEP (NEP-n) or IDE (IDE-n), or engineered secreted forms of NEP (NEP-s) or IDE (IDE-s). In a six-week study, immunohistochemistry staining for total Aβ was significantly decreased in animals receiving the NEP-n and NEP-s but not for IDE-n or IDE-s in either the hippocampus or cortex. Congo red staining followed a similar trend revealing significant decreases in the hippocampus and the cortex for NEP-n and NEP-s treatment groups. Our results indicate that while rAAV-IDE does not have the same therapeutic potential as rAAV-NEP, rAAV-NEP-s and NEP-n are effective at reducing amyloid loads, and both of these vectors continue to have significant effects nine months post-injection. As such, they may be considered reasonable candidates for gene therapy trials in AD.
Effects of C-phycocyanin (C-PC), the active component of Spirulina platensis water extract on the expressions of N-methyl D-aspartate receptor subunit 2B (NR2B), tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β), and cyclooxygenase type 2 (COX-2) genes in the cochlea and inferior colliculus (IC) of mice were evaluated after tinnitus was induced by intraperitoneal injection of salicylate. The results showed that 4-day salicylate treatment (unlike 4-day saline treatment) caused a significant increase in NR2B, TNF-α, and IL-1β mRNAs expression in the cochlea and IC. On the other hand, dietary supplementation with C-PC or Spirulina platensis water extract significantly reduced the salicylate-induced tinnitus and down-regulated the mRNAs expression of NR2B, TNF-α, IL-1β mRNAs, and COX-2 genes in the cochlea and IC of mice. The changes of protein expression levels were generally correlated with those of mRNAs expression levels in the IC for above genes.
Despite promising early results, clinical trials involving the continuous delivery of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHuGDNF) into the putamen for the treatment of Parkinson's disease have shown evidence of poor distribution and toxicity due to point-source accumulation. Convection-enhanced delivery (CED) has the potential to facilitate more widespread and clinically effective drug distribution.
We investigated acute CED of r-metHuGDNF into the striatum of normal rats in order to assess tissue clearance, toxicity (neuron loss, gliosis, microglial activation, and decreases in synaptophysin), synaptogenesis and neurite-outgrowth. We investigated a range of clinically relevant infused concentrations (0.1, 0.2, 0.6 and 1.0 µg/µL) and time points (2 and 4 weeks) in order to rationalise a dosing regimen suitable for clinical translation.
Two weeks after single dose CED, r-metHuGDNF was below the limit of detection by ELISA but detectable by immunohistochemistry when infused at low concentrations (0.1 and 0.2 µg/µL). At these concentrations, there was no associated neuronal loss (neuronal nuclei, NeuN, immunohistochemistry) or synaptic toxicity (synaptophysin ELISA). CED at an infused concentration of 0.2 µg/µL was associated with a significant increase in synaptogenesis (p<0.01). In contrast, high concentrations of r-metHuGDNF (above 0.6 µg/µL) were associated with neuronal and synaptic toxicity (p<0.01). Markers for gliosis (glial fibrillary acidic protein, GFAP) and microglia (ionized calcium-binding adapter molecule 1, Iba1) were restricted to the needle track and the presence of microglia had diminished by 4 weeks post-infusion. No change in neurite outgrowth (Growth associated protein 43, GAP43, mRNA) compared to artificial cerebral spinal fluid (aCSF) control was observed with any infused concentration.
The results of this study suggest that acute CED of low concentrations of GDNF, with dosing intervals determined by tissue clearance, has most potential for effective clinical translation by optimising distribution and minimising the risk of toxic accumulation.
Inflammation is an umbrella feature of ageing. It is present in the aged retina and many retinal diseases including age-related macular degeneration (AMD). In ageing and in AMD mitochondrial function declines. In normal ageing this can be manipulated by brief exposure to 670 nm light on the retina, which increases mitochondrial membrane potential and reduces inflammation. Here we ask if 670 nm exposure has the same ability in an aged mouse model of AMD, the complement factor H knockout (CFH−/−) where inflammation is a key feature. Further, we ask whether this occurs when 670 nm is delivered briefly in environmental lighting rather than directly focussed on the retina. Mice were exposed to 670 nm for 6 minutes twice a day for 14 days in the form of supplemented environmental light. Exposed animals had significant increase in cytochrome c oxidase (COX), which is a mitochondrial enzyme regulating oxidative phosphorylation.There was a significant reduction in complement component C3, an inflammatory marker in the outer retina. Vimetin and glial fibrillary acidic protein (GFAP) expression, which reflect retinal stress in Muller glia, were also significantly down regulated. There were also significant changes in outer retinal macrophage morphology. However, amyloid beta (Aβ) load, which also increases with age in the outer retina and is pro-inflammatory, did not change. Hence, 670 nm is effective in reducing inflammation probably via COX activation in mice with a genotype similar to that in 50% of AMD patients even when brief exposures are delivered via environmental lighting. Further, inflammation can be reduced independent of Aβ. The efficacy revealed here supports current early stage clinical trials of 670 nm in AMD patients.
The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (ChmFlox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the ChmFlox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.
Retinitis pigmentosa (RP) relates to a group of hereditary neurodegenerative diseases of the retina. On the cellular level, RP results in the primary death of rod photoreceptors, caused by rod-specific mutations, followed by a secondary degeneration of genetically normal cones. Different mechanisms may influence the spread of cell death from one photoreceptor type to the other. As one of these mechanisms a gap junction-mediated bystander effect was proposed, i.e., toxic molecules generated in dying rods and propagating through gap junctions induce the death of healthy cone photoreceptors. We investigated whether disruption of rod-cone coupling can prevent secondary cone death and reduce the spread of degeneration. We tested this hypothesis in two different mouse models for retinal degeneration (rhodopsin knockout and rd1) by crossbreeding them with connexin36-deficient mice as connexin36 represents the gap junction protein on the cone side and lack thereof most likely disrupts rod-cone coupling. Using immunohistochemistry, we compared the progress of cone degeneration between connexin36-deficient mouse mutants and their connexin36-expressing littermates at different ages and assessed the accompanied morphological changes during the onset (rhodopsin knockout) and later stages of secondary cone death (rd1 mutants). Connexin36-deficient mouse mutants showed the same time course of cone degeneration and the same morphological changes in second order neurons as their connexin36-expressing littermates. Thus, our results indicate that disruption of connexin36-mediated rod-cone coupling does not stop, delay or spatially restrict secondary cone degeneration and suggest that the gap junction-mediated bystander effect does not contribute to the progression of RP.