calpain and Retinitis-Pigmentosa

Retinitis pigmentosa (RP) is a group of inherited disorders involving the photoreceptors of the retina and can lead to visual loss. There has been tremendous progress in the delineation of the biochemical and molecular basis of RP. Reactive oxygen species, calcium-calpain activation, and lipid peroxidation are known to be involved in the initiation of photoreceptor cell death, but the precise mechanisms of this process remain unknown. Heat shock protein 70 (HSP70) has been shown to function as a chaperone molecule that protects cells against environmental and physiological stresses. However, there are a few reports showing the role of HSP70 in photoreceptor cell death. Recently, we found that the production of 4-hydroxy-2-noneral caused the calpain-dependent cleavage of carbonylated HSP70 prior to photoreceptor cell death in RP model mice. Furthermore, HSP70 inducers, such as valproic acid and geranylgeranylacetone attenuated photoreceptor cell death. HSP70 inducers may be considered as candidate therapeutic agents for RP.

Topics: Animals; Calpain; Cell Death; Disease Models, Animal; HSP70 Heat-Shock Proteins; Humans; Methylnitrosourea; Retinitis Pigmentosa

Topics: Apoptosis; Blood-Retinal Barrier; Calpain; Humans; Macular Degeneration; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa

Photoreceptor cell death is the major hallmark of a group of human inherited retinal degenerations commonly referred to as retinitis pigmentosa (RP). Although the causative genetic mutations are often known, the mechanisms leading to photoreceptor degeneration remain poorly defined. Previous research work has focused on apoptosis, but recent evidence suggests that photoreceptor cell death may result primarily from non-apoptotic mechanisms independently of AP1 or p53 transcription factor activity, Bcl proteins, caspases, or cytochrome c release. This review briefly describes some animal models used for studies of retinal degeneration, with particular focus on the rd1 mouse. After outlining the major features of different cell death mechanisms in general, we then compare them with results obtained in retinal degeneration models, where photoreceptor cell death appears to be governed by, among other things, changes in cyclic nucleotide metabolism, downregulation of the transcription factor CREB, and excessive activation of calpain and PARP. Based on recent experimental evidence, we propose a putative non-apoptotic molecular pathway for photoreceptor cell death in the rd1 retina. The notion that inherited photoreceptor cell death is driven by non-apoptotic mechanisms may provide new ideas for future treatment of RP.

Topics: Animals; Calcium; Calpain; Cell Death; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; DNA Fragmentation; Endoplasmic Reticulum; Humans; Mice; Nucleotides, Cyclic; Oxidative Stress; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa; Transcription Factors

FAM161A mutations are the most common cause of inherited retinal degenerations in Israel. We generated a knockout (KO) mouse model, Fam161a

Topics: Animals; Calpain; Disease Models, Animal; Electroretinography; Eye Proteins; Frameshift Mutation; Humans; Lac Operon; Mice; Mice, Knockout; Retina; Retinal Degeneration; Retinitis Pigmentosa; Tomography, Optical Coherence; Visual Acuity

Autosomal dominant retinitis pigmentosa (adRP) is mainly caused by mutations responsible for rhodopsin (RHO) misfolding. Although it was previously proved that unfolded RHO is retained into the endoplasmatic reticulum (ER) eliciting ER-stress, consequent mechanisms underlying photoreceptor degeneration need to be further clarified. Several animal models of RHO mutants have been developed for this purpose and for development of neuroprotective treatments. Here, we compared two of the most used models of adRP, the P23H mutant RHO transgenic and knock-in mouse models, in order to define which are their limits and potentials. Although they were largely used, the differences on the activation of the cell death pathways occurring in these two models still remain to be fully characterized. We present data proving that activation of calpains is a mechanism of cell death shared by both models and that molecules targeting calpains are neuroprotective. Conversely, the role of ER-stress contribution to cell death appears to be divergent and remains controversial.

Topics: Animals; Calpain; Cell Death; Disease Models, Animal; Endoplasmic Reticulum Stress; Gene Knock-In Techniques; Mice; Mice, Transgenic; Protein Folding; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin

Retinal degenerative diseases, such as retinitis pigmentosa, are characterized by night blindness and peripheral vision loss caused by the slowly progressive loss of photoreceptor cells. A comprehensive molecular mechanism of the photoreceptor cell death remains unclear. We previously reported that heat shock protein 70 (HSP70), which has a protective effect on neuronal cells, was cleaved by a calcium-dependent protease, calpain, in N-methyl-N-nitrosourea (MNU)-treated mice retina. Carbonylated HSP70 is much more vulnerable than noncarbonylated HSP70 to calpain cleavage. However, it was not known whether protein carbonylation occurs in MNU-treated mice retina. In this study, we clearly show protein carbonylation-dependent photoreceptor cell death induced by MNU in mice. Therefore, protein carbonylation and subsequent calpain-dependent cleavage of HSP70 are key events in MNU-mediated photoreceptor cell death. Our data provide a comprehensive molecular mechanism of the photoreceptor cell death.

Topics: Aldehydes; Animals; Calpain; Cell Death; Disease Models, Animal; Eye Proteins; HSP70 Heat-Shock Proteins; Injections, Intraperitoneal; Male; Methylnitrosourea; Mice; Mice, Inbred C57BL; Models, Molecular; Oxidative Stress; Protein Carbonylation; Retina; Retinal Degeneration; Retinitis Pigmentosa

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calcium-Binding Proteins; Calpain; Disease Models, Animal; Mice; Mutation; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin

Retinitis pigmentosa is an inherited blinding disorder characterized by progressive degeneration and loss of photoreceptors. The exact mechanism of degeneration and cell death of photoreceptors is not known, but is thought to involve disturbed Ca2+-signaling. Ca2+ can enter the photoreceptor cell via outer segment cyclic nucleotide-gated (CNG) channels or synaptic Cav1.4 L-type voltage-gated calcium channels (VGCC). Previously, we have shown that genetic ablation of the Cngb1 gene encoding the B subunit of the rod CNG channel delays the fast progressing degeneration in the rd1 mutant mouse model of retinitis pigmentosa. In this study, we crossbred rd1 mice with the Cacna1f-deficient mouse lacking the Cav1.4 α1 subunit of the L-type VGCC. Longitudinal in vivo examinations of photoreceptor layer thickness by optical coherence tomography revealed a significant, but not sustained delay of retinal degeneration in Cacna1f x rd1 double mutant mice compared to rd1 mice. This was accompanied by a reduction of TUNEL positive cells in the early phase of rod degeneration. Remarkably, Cacna1f x rd1 double mutant mice displayed a strong decrease in the activation of the Ca2+-dependent protease calpain during photoreceptor loss. Our results show that genetic deletion of the synaptic Cav1.4 L-type VGCCs impairs calpain activation and leads to a short-term preservation of photoreceptors in the rd1 mouse.

Topics: Animals; Calcium; Calcium Channels; Calcium Channels, L-Type; Calpain; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Mice; Mice, Knockout; Mutation; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Tomography, Optical Coherence

Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions.

Topics: Animals; Calpain; Caspases; Cell Survival; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Eye Proteins; Humans; Mice; Point Mutation; Retina; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases characterized by the progressive photoreceptors apoptosis. The N-Methyl- N-nitrosourea (MNU) is an alkylating toxicant which could induce photoreceptor apoptosis resembling that of the hereditary RP. However, the detailed process pattern of this degeneration remains poorly characterized. We systemically explored the topography of the photoreceptor degeneration in the MNU treated mouse, and related these spatial data with the time-dependent characteristics of retinal pathology. These temporal topographic data delineated sequential scenes of the progressive photoreceptor degeneration in the MNU treated retinas: focal photoreceptors showed different vulnerabilities to the MNU toxicity and displayed a distinctive spatial- and time-dependent progression. Moreover, the positional asymmetry between the retinal quadrants firstly provided instructive information about the unique toxicology properties of the MNU. Further mechanism study suggested that the up-regulation of Bax and Calpain-2, rather than the Caspase-3, should be responsible for the asymmetry in the MNU induced photoreceptor degeneration. Together with the comparative sensitivities to the neurotoxicity of MNU between two photoreceptor populations, these topographic data would facilitate the standardization of analytic parameters related to the MNU induced RP model, and enhance its application in the therapeutic explorations of human RP.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calpain; Caspase 3; Gene Expression Regulation; Humans; Methylnitrosourea; Mice; Photoreceptor Cells; Retina; Retinal Degeneration; Retinitis Pigmentosa

We previously reported activation of the unfolded protein response (UPR) in P23H rhodopsin (RHO) retinas with autosomal dominant retinitis pigmentosa (ADRP). Knowing that the UPR can trigger Ca(2+) release from the endoplasmic reticulum and regulate cellular signaling we examined the level of Ca(2+)-regulated proteins. We also looked for changes in the expression of Bcl2 family proteins, autophagy proteins and the mTOR/AKT pathways, as well as for the induction of mitochondria-associated apoptosis in the P23H RHO retina. Our data demonstrated that the elevation of calpain and caspase-12 activity was concomitantly observed with a decrease in the BCL2-XL/BAX ratio and an increase in mTor levels in the P23H-3 RHO retina suggesting a vulnerability of P23H RHO photoreceptors to apoptosis. The translocation of BAX to the mitochondria, as well as the release of cytochrome C and AIF into the cytosol supports this conclusion and indicates the involvement of mitochondria-induced apoptosis in the progression of ADRP. The level of autophagy proteins in general was found to be decreased in the P21-P30 P23H RHO retina. Injections of rapamycin, however, protected the P23H RHO rod photoreceptors from experiencing physiological decline. Despite this fact, the downregulation of mTOR did not alter the level of autophagy proteins. Our results imply that in addition to activation of the UPR during ADRP progression, photoreceptors also experience alterations in major proapoptotic pathways.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Calcium; Calpain; Caspase 12; Cytochromes c; Humans; Mitochondria; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa; Rhodopsin; Signal Transduction; TOR Serine-Threonine Kinases; Up-Regulation

The process of photoreceptor cell death in retinitis pigmentosa is still not well characterized, and identification of common mechanisms will be instrumental for development of therapeutic strategies. Here we investigated activation of Bax in rd1, P23H transgenic, and Rho knockout retinas.. Bax activation was evaluated by immunofluorescence using anti-activated Bax-specific antibodies and by Western blotting on mitochondrial protein extracts. Knockdown of cathepsin D, calpain 1, and calpain 2 was achieved by short hairpin RNA (shRNA) delivery in rd1 mutant photoreceptors cells differentiated from retinal neurospheres. The mechanism of Bax activation through calpains was evaluated in vivo by intravitreal injection of calpastatin.. We defined activation and mitochondrial localization of Bax as well as activation of calpains and cathepsin D in the three models of retinitis pigmentosa. Taking advantage of an in vitro culture system for rd1 mutant photoreceptors, we unraveled the mechanism of Bax activation. We demonstrated that calpain 1 and cathepsin D contributed to activation of Bax and to apoptosis-inducing factor (Aif) nuclear translocation. In vivo interference with calpain activity blocks Bax activation in the rd1 and Rho knockout retinas and reduces activation in the P23H transgenic retina.. Activation of Bax was observed in all three models of retinitis pigmentosa and leads to neurodamage by localization at the mitochondrion. Our data suggest that Bax can be envisaged as one of the promising target molecules for restraining photoreceptor degeneration.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium-Binding Proteins; Calpain; Cells, Cultured; Cysteine Proteinase Inhibitors; Disease Models, Animal; In Situ Nick-End Labeling; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mutation; Photoreceptor Cells, Vertebrate; Real-Time Polymerase Chain Reaction; Retinitis Pigmentosa; RNA; Transcriptional Activation

Inherited retinal degenerations affecting both rod and cone photoreceptors constitute one of the causes of incurable blindness in the developed world. Cyclic guanosine monophosphate (cGMP) is crucial in the phototransduction and, mutations in genes related to its metabolism are responsible for different retinal dystrophies. cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4-5% of the retinitis pigmentosa, a rare form of retinal degeneration. The aim of this study was to evaluate whether pharmacological PDE6 inhibition induced retinal degeneration in cone-enriched cultures of porcine retina similar to that found in murine models. PDE6 inhibition was induced in cone-enriched retinal explants from pigs by Zaprinast. PDE6 inhibition induced cGMP accumulation and triggered retinal degeneration, as determined by TUNEL assay. Western blot analysis and immunostaining indicated that degeneration was accompanied by caspase-3, calpain-2 activation and poly (ADP-ribose) accumulation. Oxidative stress markers, total antioxidant capacity, thiobarbituric acid reactive substances (TBARS) and nitric oxide measurements revealed the presence of oxidative damage. Elevated TNF-alpha and IL-6, as determined by enzyme immunoassay, were also found in cone-enriched retinal explants treated with Zaprinast. Our study suggests that this ex vivo model of retinal degeneration in porcine retina could be an alternative model for therapeutic research into the mechanisms of photoreceptor death in cone-related diseases, thus replacing or reducing animal experiments.

Topics: Animals; Apoptosis; Calpain; Caspase 3; Cyclic GMP; In Situ Nick-End Labeling; Organ Culture Techniques; Oxidative Stress; Phosphodiesterase Inhibitors; Purinones; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Swine; Swine, Miniature

Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-µCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated Royal College of Surgeons rats, one of the animal models of RP. Because of the high rate of rhodopsin mutations in RP patients, the present study was performed to confirm the protective effects of Tat-µCL against retinal degeneration in rhodopsin transgenic S334ter and P23H rats. We examined the effects of intravitreal injection or topical application of the peptide on retinal degeneration in S334ter and P23H rats by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electroretinogram (ERG), immunohistochemistry for AIF, and histological staining. In S334ter rats, we found that intravitreal injection or topical application of the peptide prevented photoreceptor cell death from postnatal (PN) 15 to 18 days, the time of early-stage retinal degeneration. Topical application of the peptide also delayed attenuation of ERG responses from PN 28 to 56 days. In P23H rats, topical application of the peptide protected against photoreceptor cell death and nuclear translocation of AIF on PN 30, 40, and 50 days, as the primary stages of degeneration. We observed that topical application of the peptide inhibited the thinning of the outer nuclear layer and delayed ERG attenuations from PN 30 to 90 days. Our results demonstrate that the mitochondrial μ-calpain and AIF pathway is involved in early-stage retinal degeneration in rhodopsin transgenic S334ter and P23H rats, and inhibition of this pathway shows curative potential for rhodopsin mutation-caused RP.

Topics: Animals; Apoptosis Inducing Factor; Calpain; Cell Death; Cell Nucleus; Disease Models, Animal; Electroretinography; Gene Expression Regulation; In Situ Nick-End Labeling; Intravitreal Injections; Mitochondria; Mitochondrial Proteins; Mutation; Ophthalmic Solutions; Peptides; Photoreceptor Cells, Vertebrate; Protein Transport; Rats; Rats, Transgenic; Retinitis Pigmentosa; Rhodopsin; Signal Transduction

Activations of mitochondrial calpains cause apoptosis-inducing factor-dependent apoptosis of retinal photoreceptor cells in the Royal College of Surgeons (RCS) rat, an animal model of retinitis pigmentosa. In the present study, we attempted to develop specific inhibitors of mitochondrial calpains that would prevent the retinal degeneration. We examined the inhibitory potency of 20-mer peptides of the m-calpain for mitochondrial calpains activity, determined the inhibitory regions, and conjugated the cell-penetrating peptides (CPP). The cytotoxicity and delivery of the peptide was evaluated using mouse photoreceptor-derived 661W cells. After intravitreal injection of the peptide in RCS rats, we examined the peptide delivery to the retina, photoreceptor cell death numbers, responses of the electroretinogram (ERG), concentrations of intracellular ATP, and changes of retinal morphology. Results showed that one of the peptides inhibited the activity of the mitochondrial m-calpain. The HIV-1 tat-conjugated m-calpain peptide, HIV-Nm, could preserve the inhibitory potency of the mitochondrial m-calpain, and penetrate into the 661W cells. While intravitreal injection of HIV-Nm made it possible to deliver to the retina, it did not prevent photoreceptor cell death. Furthermore, it caused the ERG attenuation and the decrease in the intracellular ATP only a day after the injection. Although HIV-Nm did not cause histological change of the retina after 1 or 2 days of the administration, the morphological abnormality of the retina was observed after 3-14 days. Our results demonstrated that HIV-Nm failed to prevent the photoreceptor cell death, but rather caused the attenuation of ERG response and the decrease of ATP.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Apoptosis; Apoptosis Inducing Factor; Calpain; Electroretinography; Glycoproteins; Mitochondria; Molecular Sequence Data; Peptides; Photoreceptor Cells, Vertebrate; Protein Transport; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Retinal Degeneration; Retinitis Pigmentosa; Sequence Alignment; tat Gene Products, Human Immunodeficiency Virus

Retinitis pigmentosa (RP) is a genetically heterogenous group of inherited retinal degenerative diseases resulting from photoreceptor cell death and affecting >1 million persons globally. Although oxidative stress has been implicated in the pathogenesis of RP, the mechanisms by which oxidative stress mediates photoreceptor cell death are largely unknown. Here, we show that oxidation of nucleic acids is a key component in the initiation of death-signaling pathways in rd10 mice, a model of RP. Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) increased in photoreceptor cells, and especially within their nuclei, in rd10 mice as well as in Royal College of Surgeons rats, another model of RP caused by different genetic mutations. Vitreous samples from humans with RP contained higher levels of 8-oxo-dG excreted than samples from nondegenerative controls. Transgenic overexpression of human MutT homolog-1, which hydrolyzes oxidized purine nucleoside triphosphates in the nucleotide pool, significantly attenuated 8-oxo-dG accumulation in nuclear DNA and photoreceptor cell death in rd10 mice, in addition to suppressing DNA single-strand break formation, poly(ADP-ribose) polymerase activation, and nuclear translocation of apoptosis-inducing factor. These findings indicate that oxidative DNA damage is an important process for the triggering of photoreceptor cell death in rd10 mice and suggest that stimulation of DNA repair enzymes may be a novel therapeutic approach to attenuate photoreceptor cell loss in RP.

Topics: Animals; Apoptosis Inducing Factor; Calpain; Cell Death; Cell Nucleus; Disease Models, Animal; DNA Breaks, Single-Stranded; DNA Damage; DNA Repair Enzymes; Enzyme Activation; Humans; Inheritance Patterns; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxidation-Reduction; Phosphoric Monoester Hydrolases; Photoreceptor Cells, Vertebrate; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Transport; Rats; Retinal Degeneration; Retinitis Pigmentosa; Signal Transduction

Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.

Topics: Amino Acid Sequence; Animals; Apoptosis; Calpain; Disease Models, Animal; Humans; Intravitreal Injections; Mitochondria, Liver; Molecular Sequence Data; Ophthalmic Solutions; Peptides; Photoreceptor Cells; Protein Conformation; Protein Structure, Tertiary; Rats; Retinitis Pigmentosa; tat Gene Products, Human Immunodeficiency Virus

Photoreceptor degeneration is the hallmark of a group of inherited blinding diseases collectively termed retinitis pigmentosa (RP); a major cause of blindness in humans. RP is at present untreatable and the underlying neurodegenerative mechanisms are largely unknown, even though the genetic causes are often established. The activation of calpain-type proteases may play an important role in cell death in various neuronal tissues, including the retina. We therefore tested the efficacy of two different calpain inhibitors in preventing cell death in the retinal degeneration (rd1) human homologous mouse model for RP. Pharmacological inhibition of calpain activity in rd1 organotypic retinal explants had ambiguous effects on photoreceptor viability. Calpain inhibitor XI had protective effects when applied for short periods of time (16 h) but demonstrated substantial levels of toxicity in both wild-type and rd1 retina when used over several days. In contrast, the highly specific calpain inhibitor calpastatin peptide reduced photoreceptor cell death in vitro after both short and prolonged exposure, an effect that was also evident after in vivo application via intravitreal injection. These findings highlight the importance of calpain activation for photoreceptor cell death but also for photoreceptor survival and propose the use of highly specific calpain inhibitors to prevent or delay RP.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Death; Glycoproteins; Humans; Mice; Mice, Inbred C3H; Mice, Transgenic; Organ Culture Techniques; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinitis Pigmentosa

Degenerative retinopathies, including age-related macular degeneration, diabetic retinopathy, and hereditary retinal disorders--major causes of world blindness--are potentially treatable by using low-molecular weight neuroprotective, antiapoptotic, or antineovascular drugs. These agents are, however, not in current systemic use owing to, among other factors, their inability to passively diffuse across the microvasculature of the retina because of the presence of the inner blood-retina barrier (iBRB). Moreover, preclinical assessment of the efficacies of new formulations in the treatment of such conditions is similarly compromised. We describe here an experimental process for RNAi-mediated, size-selective, transient, and reversible modulation of the iBRB in mice to molecules up to 800 Da by suppression of transcripts encoding claudin-5, a protein component of the tight junctions of the inner retinal vasculature. MRI produced no evidence indicative of brain or retinal edema, and the process resulted in minimal disturbance of global transcriptional patterns analyzed in neuronal tissue. We show that visual function can be improved in IMPDH1(-/-) mice, a model of autosomal recessive retinitis pigmentosa, and that the rate of photoreceptor cell death can be reduced in a model of light-induced retinal degeneration by systemic drug delivery after reversible barrier opening. These findings provide a platform for high-throughput drug screening in models of retinal degeneration, and they ultimately could result in the development of a novel "humanized" approach to therapy for conditions with little or no current forms of treatment.

Topics: Animals; Blood-Retinal Barrier; Calpain; Claudin-5; Cysteine Proteinase Inhibitors; Disease Models, Animal; Drug Delivery Systems; Electroretinography; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Magnetic Resonance Imaging; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oligopeptides; Retina; Retinitis Pigmentosa; RNA Interference; RNA, Small Interfering