Analysis of six candidate genes as potential modifiers of disease expression in canine XLPRA1, a model for human X-linked retinitis pigmentosa 3

Purpose: Canine X-linked progressive retinal atrophy (XLPRA) is caused by mutations in RPGR exon ORF15, which is also a mutation hotspot in human X-linked retinitis pigmentosa 3 (RP3). The XLPRA1 form of disease has shown extensive phenotypic variability in a colony of dogs that all inherited the same mutant X-chromosome. This variability in onset and severity makes XLPRA1 a valuable model to use to identify genes influencing photoreceptors degeneration in dog and to elucidate molecular mechanisms underlying RP in its human homolog. In this study, RPGRIP1, RANBP2, NPM1, PDE6D, NPHP5, and ABCA4 genes were selected on the basis of interaction with RPGR or RPGRIP1 or their implication in related retinal diseases, and were investigated as candidate genetic modifiers of XLPRA1. Methods: A pedigree derived from an affected male dog outcrossed to unrelated normal mix bred or purebred females was used. Morphologic examination revealed phenotypic variability in the affected dogs characterized as mild, moderate, or severe. Single nucleotide polymorphisms (SNPs) and indel-containing markers spanning the entire genes were designed, based on the canine sequence and the Broad Institute SNP library, and genotyped on the pedigree. For each candidate gene, haplotypes were identified and their frequencies in severely and moderately affected dogs were compared to detect a putative correlation between a gene-specific haplotype(s), and severity level of the disease. Primers were derived from expressed sequence tags (ESTs) and predicted transcripts to assess the relative retinal expression of the six genes of interest in normal and affected retinas of different ages. Results: Four to seven haplotypes per gene were identified. None of the haplotypes of RPGRIP1, NPM1, PDE6D, NPHP5, RANBP2, and ABCA4 were found to co-segregate with the moderate or severe phenotype. No significant difference in the retinal expression levels of the candidate genes was observed between normal and affected dogs. Conclusions: The haplotype distribution of RPGRIP1, NPM1, PDE6D, NPHP5, RANBP2, and ABCA4 suggests these genes are not modifiers of the disease phenotype observed in the XLPRA1 pedigree. The RPGRORF15 stop mutation does not affect the retinal expression of these genes at the mRNA level in the pre-degenerate stage of disease, but no conclusions can be made at this time about changes that may occur at the protein level

Doxorubicin-Induced Cardiotoxicity in Collaborative Cross (CC) Mice Recapitulates Individual Cardiotoxicity in Humans.

Anthracyclines cause progressive cardiotoxicity whose ultimate severity is individual to the patient. Genetic determinants contributing to this variation are difficult to study using current mouse models. Our objective was to determine whether a spectrum of anthracycline induced cardiac disease can be elicited across 10 Collaborative Cross mouse strains given the same dose of doxorubicin. Mice from ten distinct strains were given 5 mg/kg of doxorubicin intravenously once weekly for 5 weeks (total 25 mg/kg). Mice were killed at acute or chronic timepoints. Body weight was assessed weekly, followed by terminal complete blood count, pathology and a panel of biomarkers. Linear models were fit to assess effects of treatment, sex, and sex-by-treatment interactions for each timepoint. Impaired growth and cardiac pathology occurred across all strains. Severity of these varied by strain and sex, with greater severity in males. Cardiac troponin I and myosin light chain 3 demonstrated strain- and sex-specific elevations in the acute phase with subsequent decline despite ongoing progression of cardiac disease. Acute phase cardiac troponin I levels predicted the ultimate severity of cardiac pathology poorly, whereas myosin light chain 3 levels predicted the extent of chronic cardiac injury in males. Strain- and sex-dependent renal toxicity was evident. Regenerative anemia manifested during the acute period. We confirm that variable susceptibility to doxorubicin-induced cardiotoxicity observed in humans can be modeled in a panel of CC strains. In addition, we identified a potential predictive biomarker in males. CC strains provide reproducible models to explore mechanisms contributing to individual susceptibility in humans

Mutation of A DNA Repair Enzyme Causes Lupus in Mice

A replication study of a previous genome-wide association study (GWAS) suggested that a SNP linked to the POLβ gene is associated with systemic lupus erythematosus (SLE). This SNP is correlated with decreased expression of Pol β, a key enzyme in the base excision repair (BER) pathway. To determine whether decreased Pol β activity results in SLE, we constructed a mouse model of POLβ that encodes an enzyme with slow DNA polymerase activity. We show that mice expressing this hypomorphic POLβ allele develop an autoimmune pathology that strongly resembles SLE. Of note, the mutant mice have shorter immunoglobulin heavy-chain junctions and somatic hypermutation is dramatically increased. These results demonstrate that decreased Pol β activity during the generation of immune diversity leads to lupus-like disease in mice, and suggest that decreased expression of Pol β in humans is an underlying cause of SLE

Safety in Nonhuman Primates of Ocular AAV2-\u3cem\u3eRPE65\u3c/em\u3e, a Candidate Treatment for Blindness in Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV- RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2. RPE65 in RPE65 -mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vector-related toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2. RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2. RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina

Salted roads lead to oedema and reduced locomotor function in amphibian populations

Human activities have caused massive losses of natural populations across the globe. Like many groups, amphibians have experienced substantial declines worldwide, driven by environmental changes such as habitat conversion, pollution, and disease emergence. Each of these drivers is often found in close association with the presence of roads. Here we report a novel consequence of roads affecting an amphibian native to much of North America, the wood frog (Rana sylvatica). Across 38 populations distributed from southern to central New England, we found that adult wood frogs living adjacent to roads had higher incidence and severity of oedema (indicated by obvious bloating caused by subcutaneous fluid accumulation) during the breeding season than frogs living away from the influence of roads. This effect was best explained by increased conductivity of breeding ponds, prob-ably caused by runoff pollution from road salt used for de-icing. Oedema severity was negatively correlated with locomotor performance in more northerly populations. Interestingly, northern populations experience more intense winters, which tends to result in more de-icing salt runoff and increased energetic demands associated with overwintering cryoprotection needs. Thus, this emerging consequence of roads appears to impose potential fitness costs associated with locomotion, and these effects might be most impactful on populations living in regions where de-icing is most intense.Together, our findings reveal a novel set of impacts of roads and runoff pollution on wood frog physiology and performance, which seem likely to contribute to population decline. Given the global prevalence of roads and increasing salinisation of freshwater habitats, oedema and related impacts could be widespread consequences faced by amphibian populations across much of the planet's temperate zonesThis work was supported by Mianus River Gorge Preserve, Elm City Innovation Collaborative, Yale Institute for Biospheric Studies, EEES Graduate fellowship and Cramer funds, Guarini School of Graduate and Advanced Studies McCulloch Fellowship, CAPES graduate fellowship (SwB 13442/13-9), the Margarita Salas Fellowship, and the National Science Foundation (DEB #1011335, DEB #1655092).Peer reviewe

Retinal pathology of canine X-linked progressive retinal atrophy, the locus homologue of RP3

PURPOSE. To describe the course of photoreceptor disease in canine X-linked retinal degeneration. METHODS. Retinas from 55 dogs (44 males, 8 carrier females, 3 homozygous females) were obtained by enucleation under general anesthesia. After fixation and dehydration, tissues were embedded in epoxy resin, sectioned at 1 m for light microscopy and stained with azure II/methylene blue and a paraphenylenediamine counterstain. For electron microscopy, regions identified by light microscopy were selected and cut at 60 nm. Sections were stained with uranyl acetate-lead citrate. Electroretinography from an additional group of normal males, affected males, and carrier females was performed and the rod and cone responses evaluated. RESULTS. The earliest lesion detectable by electron microscopy was vesiculation of rod discs, followed by disruption of outer segments and death of rods. Loss of cones and progressive atrophy of inner retinal layers followed. Lesions were most severe in the peripheral retina and advanced toward the optic disc with disease progression. Significant variation in disease severity was present in males despite the presence of the same disease allele in all affected dogs. Carrier females displayed generalized reduction in photoreceptor density as well as multifocal areas of complete rod loss. The electroretinogram (ERG) findings were compatible with the histopathologic abnormalities. Homozygous females had lesions similar to those seen in affected males. CONCLUSIONS. X-linked retinal degeneration is characterized by initial degeneration of rod photoreceptors, followed by loss of cones and progressive atrophy of the inner retina. Carrier females display a phenotype consistent with random X-chromosome inactivation. Variation in genetic background may alter expression of the disease allele in affected animals, thus accounting for variation in phenotypic expression of the disease. (Invest Ophthalmol Vis Sci. 1999;40:3292-3304) R etinitis pigmentosa (RP) describes a group of genetically heterogeneous inherited retinal disorders characterized by progressive photoreceptor disease, degeneration, and cell death. RP may be inherited in autosomal recessive, dominant or X-linked patterns. Of these forms, the X-linked form is the least prevalent (16%-33% of all RP cases) 1,2 but the most severe in early age of onset and rate of progression. 3 Approximately 70% of X-linked retinitis pigmentosa (XLRP) cases map to one of two loci on the X chromosome: RP2 or RP3. 4 RP3 is the predominant form of XLRP and has been localized to a 1-cM region at Xp21. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for 20% to 30% of RP3 cases

Mapping of X-linked Progressive Retinal Atrophy (XLPRA), the Canine Homolog of Retinitis Pigmentosa 3 (RP3

X-linked progressive retinal atrophy (XLPRA) in the Siberian husky dog is a naturally occurring X-linked retinopathy closely resembling X-linked retinitis pigmentosa (XLRP) in humans. In affected males, initial degeneration of rods is followed by cone degeneration and complete retinal atrophy; carrier females have random patches of rod degeneration consistent with random X chromosome inactivation. By typing the XLPRA pedigree with five intragenic markers [dystrophin, retinitis pigmentosa GTPase regulator (RPGR), tissue inhibitor of metalloproteinases 1, androgen receptor and factor IX], we established a linkage map of the canine X chromosome, and confirmed that the order of these five genes is identical to that on the human X. XLPRA was tightly linked to an intragenic RPGR polymorphism (LOD 11.7, zero recombination), thus confirming locus homology with RP3. We cloned the full-length canine RPGR cDNA and three additional splice variants. No disease-causing mutation was found in the RPGR-coding sequence of the four splice variants characterized, a finding similar to ~80% of human XLRP patients whose disease maps to the RP3 locus. In addition, there were no significant differences in the proportional expression of each splice variant in normal and pre-degenerate XLPRA-affected retina. Expression of all RPGR splice variants increased later in the disease, when retinas were undergoing active degeneration. The results provide further evidence of cross-species retention of a complex splicing pattern in the 3′ portion of RPGR, the functional significance of which is unknown. In addition, the possibility of another disease locus in the RP3 region is supported. cAROLIN

Established patterns of animal study design undermine translation of disease-modifying therapies for Parkinson’s disease

<div><p>Translation of disease-modifying therapies in neurodegenerative disease has been disappointing. Parkinson’s disease (PD) was used to compare patterns of preclinical study design for symptomatic and potentially disease-modifying interventions. We examined the relationship of model, intervention type and timing, outcomes and outcome measures in 543 animal and human studies (1973–2015) across a contemporary cohort of animal and human interventional studies (n = 445), animal studies for approved interventions (n = 28), animal and human studies for those that failed to translate (n = 70). Detailed study design data were collected for 216 studies in non-human primate (NHP) and rodent toxin-induced models. Species-specific patterns of study design prevailed regardless of whether interventions were symptomatic or potentially disease-modifying. In humans and NHPs, interventions were typically given to both sexes well after the PD phenotype was established, and clinical outcome measures were collected at single (symptomatic) or multiple (disease-modifying) time-points. In rodents, interventions often preceded induction of the model, acute toxic protocols were common, usually given to young males, clinical outcome measures were used less commonly, and outcomes were less commonly assessed at multiple time points. These patterns were more prevalent in mice than rats. In contrast, study design factors such as randomization and blinding did not differ appreciably across symptomatic and disease-modifying intervention categories. The translational gap for potentially disease-modifying interventions in PD in part results from study designs, particularly in mice, that fail to model the progressive nature and relatively late intervention characteristic of PD, or that anchor mechanistic and neuropathologic data to longitudinal clinical outcomes. Even if measures to improve reproducibility are broadly adopted, perpetuation of these norms will continue to impede effective translation.</p></div

Outcomes by species and type of intervention (n = 445 studies).

<p>Outcomes by species and type of intervention (n = 445 studies).</p