Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues.

Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted.

T Cell Lymphoma and Leukemia in Severe Combined Immunodeficiency Pigs following Bone Marrow Transplantation: A Case Report.

After the discovery of naturally occurring severe combined immunodeficiency (SCID) within a selection line of pigs at Iowa State University, we found two causative mutations in the Artemis gene: haplotype 12 (ART12) and haplotype 16 (ART16). Bone marrow transplants (BMTs) were performed to create genetically SCID and phenotypically immunocompetent breeding animals to establish a SCID colony for further characterization and research utilization. Of nine original BMT transfer recipients, only four achieved successful engraftment. At approximately 11 months of age, both animals homozygous for the ART16 mutation were diagnosed with T cell lymphoma. One of these ART16/ART16 recipients was a male who received a transplant from a female sibling; the tumors in this recipient consist primarily of Y chromosome-positive cells. The other ART16/ART16 animal also presented with leukemia in addition to T cell lymphoma, while one of the ART12/ART16 compound heterozygote recipients presented with a nephroblastoma at a similar age. Human Artemis SCID patients have reported cases of lymphoma associated with a "leaky" Artemis phenotype. The naturally occurring Artemis SCID pig offers a large animal model more similar to human SCID patients and may offer a naturally occurring cancer model and provides a valuable platform for therapy development.

Evaluation of three hand-held tonometers in normal canine eyes.

Tonometry, an indirect measurement of intraocular pressure (IOP), is important for the diagnosis and management of glaucoma and uveitis. The aim of this study was to compare the performance of three hand-held tonometers in normal canine eyes. Eyes from cadavers of dogs without observable ocular disease were used to compare tonometric measurements with direct manometry over a range of 7.4-65mmHg. In vivo measurements using the three tonometers in both eyes of 12 healthy dogs were compared. All tonometers significantly underestimated manometric values both ex vivo and in vivo. One tonometer showed a small fixed bias over the range of IOP, whilst the other two tonometers had a negative proportional bias. The results of this study show that differences exist between handheld tonometers across the clinically relevant range of IOP, and that all underestimate manometric measurements.

Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor.

The GluN2B subunit of the N-methyl-d-aspartate (NMDA) receptor shows age-related declines in expression across the frontal cortex and hippocampus. This decline is strongly correlated to age-related memory declines. This study was designed to determine if increasing GluN2B subunit expression in the frontal lobe or hippocampus would improve memory in aged mice. Mice were injected bilaterally with either the GluN2B vector, containing cDNA specific for the GluN2B subunit and enhanced green fluorescent protein (eGFP); a control vector or vehicle. Spatial memory, cognitive flexibility, and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression, exhibited improved long-term spatial memory, comparable to young mice. However, memory was rescued on different days in the Morris water maze; early for hippocampal GluN2B subunit enrichment and later for the frontal lobe. A higher concentration of the GluN2B antagonist, Ro 25-6981, was required to impair long-term spatial memory in aged mice with enhanced GluN2B expression, as compared to aged controls, suggesting there was an increase in the number of GluN2B-containing NMDA receptors. In addition, hippocampal slices from aged mice with increased GluN2B subunit expression exhibited enhanced NMDA receptor-mediated excitatory post-synaptic potentials (EPSP). Treatment with Ro 25-6981 showed that a greater proportion of the NMDA receptor-mediated EPSP was due to the GluN2B subunit in these animals, as compared to aged controls. These results suggest that increasing the production of the GluN2B subunit in aged animals enhances memory and synaptic transmission. Therapies that enhance GluN2B subunit expression within the aged brain may be useful for ameliorating age-related memory declines.

Gene therapy ameliorates cardiovascular disease in dogs with mucopolysaccharidosis VII.

BACKGROUND: Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease caused by deficient beta-glucuronidase (GUSB) activity resulting in defective catabolism of glycosaminoglycans (GAGs). Cardiac disease is a major cause of death in MPS VII because of accumulation of GAGs in cardiovascular cells. Manifestations include cardiomyopathy, mitral and aortic valve thickening, and aortic root dilation and may cause death in the early months of life or may be compatible with a fairly normal lifespan. We previously reported that neonatal administration of a retroviral vector (RV) resulted in transduction of hepatocytes, which secreted GUSB into the blood and could be taken up by cells throughout the body. The goal of this study was to evaluate the effect on cardiac disease. METHODS AND RESULTS: Six MPS VII dogs were treated intravenously with an RV-expressing canine GUSB. Echocardiographic parameters, cardiovascular lesions, and biochemical parameters of these dogs were compared with those of normal and untreated MPS VII dogs. CONCLUSIONS: RV-treated dogs were markedly improved compared with untreated MPS VII dogs. Most RV-treated MPS VII dogs had mild or moderate mitral regurgitation at 4 to 5 months after birth, which improved or disappeared when evaluated at 9 to 11 and at 24 months. Similarly, mitral valve thickening present early in some animals disappeared over time, whereas aortic dilation and aortic valve thickening were absent at all times. Both myocardium and aorta had significant levels of GUSB and reduction in GAGs.

A model of mucopolysaccharidosis IIIB (Sanfilippo syndrome type IIIB): N-acetyl-alpha-D-glucosaminidase deficiency in Schipperke dogs.

Mucopolysaccharidosis III (MPS III) is characterized by lysosomal accumulation of the glycosaminoglycan (GAG) heparan sulphate (HS). In humans, the disease manifests in early childhood, and is characterized by a progressive central neuropathy leading to death in the second decade. This disease has also been described in mice (MPS IIIA and IIIB), dogs (MPS IIIA), emus (MPS IIIB) and goats (MPS IIID). We now report on dogs with naturally occurring MPS IIIB, detailing the clinical signs, diagnosis, histopathology, tissue enzymology and substrate levels. Two 3-year-old Schipperke dogs were evaluated for tremors and episodes of stumbling. Examination of the animals found signs consistent with cerebellar disease including dysmetria, hind limb ataxia and a wide-based stance with truncal swaying. There were mildly dystrophic corneas and small peripheral foci of retinal degeneration. Magnetic resonance imaging of the brain and skeletal radiographs were normal. Intracytoplasmic granules were found in the white cells of peripheral blood and cerebral spinal fluid, and in myeloid lineages in bone marrow. Electrophoresis of urinary GAGs indicated the presence of HS, while assays of cultured fibroblasts found N-acetyl-alpha-D-glucosaminidase (Naglu) activity of between 4.3% and 9.2% of normal. Owing to neurological deterioration, both dogs were euthanized, and post-mortem examinations were performed. Biochemical studies of liver and kidney from both animals demonstrated profound deficiency of Naglu activity and abnormally high GAG levels. Pathology of the brain included severe cerebellar atrophy, Purkinje cell loss, and cytoplasmic vacuolation in neurons and perithelial cells throughout the central nervous system. Pedigree analyses and Naglu levels of family members supported an autosomal recessive mode of inheritance. Using an obligate heterozygote, a breeding colony has been established to aid in understanding the pathogenesis of MPS IIIB and testing of potential therapies.

MC1R studies in dogs with melanistic mask or brindle patterns.

Black mask is a characteristic pattern in which red, yellow, tan, fawn, or brindle dogs exhibit a melanistic muzzle which may extend up onto the ears. Melanistic mask is inherited in several breeds as an autosomal dominant trait, and appears to be a fixed trait in a few breeds of dogs. A MC1R nonsense mutation, R306ter, has been shown to cause a completely red or yellow coat color in certain breeds such as Irish setters, yellow Labrador retrievers, and golden retrievers. The amino acid sequence for the melanocortin receptor 1 gene (MC1R) was examined in 17 dogs with melanistic masks from seven breeds, 19 dogs without melanistic masks, and 7 dogs in which their coat color made the mask difficult to distinguish. We also examined nine brindle dogs of four breeds, including three dogs who also had a black mask. No consistent amino acid change was observed in the brindle dogs. All dogs with a melanistic mask had at least one copy of a valine substitution for methionine at amino acid 264 (M264V) and none were homozygous for the premature stop codon (R306ter). These results suggest that black mask, but not brindle, is caused by a specific MC1R allele.

Animal models for mucopolysaccharidoses and their clinical relevance.

The mucopolysaccharidoses (MPS) are characterized by the accumulation of glycosaminoglycans (GAG) and result from the impaired function of one of 11 enzymes required for normal GAG degradation. MPS II was the first MPS to be defined clinically in humans and is caused by deficient activity of the enzyme iduronate-2-sulphatase. MPS VI was the first MPS recognized in an animal; since then, all but MPS IIIC and IX have been described as naturally occurring in animals or made by knock-out technology. As in humans, all are inherited as autosomal recessive traits, except for MPS II, which is X-linked. Most animal colonies have been established from single related heterozygous animals, making the affected offspring homozygous for the same mutant allele. Importantly, these models have disease pathology that is similar to that seen in humans, making the animals extremely valuable for the investigation of disease pathogenesis and the testing of therapies. Large animal homologues are similar to humans in natural genetic diversity, approaches to therapy and care, and the possibility of evaluating long-term effects of treatment. Therapeutic strategies for MPS include enzyme replacement therapy, heterologous bone marrow transplantation, and somatic cell gene transfer, all of which have been tested in animals with some success.

Cloning and characterization of cDNAs for a bovine (Bos taurus) Mx protein.

Mx proteins are GTPases that are stringently induced in cells from many vertebrates on exposure to type I interferons (IFNs), and expression of some Mx proteins potently inhibits replication of specific viruses. Two cDNAs encoding bovine Mx proteins were isolated from an endometrial phage library. The open reading frames (ORFs) of these two clones predict proteins of 654 (Mxl) and 648 (Mxl-a) residues. Both possess the tripartite GTPase domains, dynamin signature, and leucine zipper motifs conserved in all other Mx proteins identified. The bovine protein sequences show highest identity to ovine Mx (93%) and are substantially similar to human MxA (73%) and mouse Mx1 (63%). Based on differences between the two bovine clones in the coding and 3'-untranslated regions, it was concluded that they represent two alleles of one gene, and heterozygous and homozygous cattle were identified. Expression of Mx mRNA was rapidly induced in cultured bovine cells by treatment with IFN.

Deficiency of fucosidase results in acrosomal dysgenesis and impaired sperm maturation.

Although a variety of glycosyltransferases and glycosidases have been implicated in spermatogenesis and posttesticular sperm maturation, the biological role of these enzymes in these processes is largely unknown. We describe reproductive sequelae in a cohort of male dogs suffering from fucosidosis, a heritable lysosomal storage disorder caused by a severe deficiency of alpha-L-fucosidase. There was a reduction in the total number of sperm in the ejaculate. Only 3-5% of sperm were motile. None of the sperm were found to be morphologically normal. The predominant morphological defects observed were malformed acrosomes (56%) and retained proximal cytoplasmic droplets (92%), indicating that spermiogenesis and sperm maturation were impaired. The cytoplasm of all cellular components of the testis and excurrent ducts were vacuolated. The vacuolation resulted from enlargement of lysosomes caused by accumulation of compounds that are otherwise cleaved/degraded when lysosomal hydrolases are present normally. It is possible that impairment in spermatogenesis, particularly morphogenesis of the acrosome, is due to physical damage caused by anomalous enlargement of lysosomes. Although an unambiguous causal relationship could not be established, it is evident from the available information that the derangement in events associated with epididymal sperm maturation, namely acquisition of motility and shedding of the cytoplasmic droplet, is likely due to lack of fucosidase leading to impaired sperm membrane modification. This heritable condition in dogs may serve as a spontaneously occurring knock-out model for further elucidating the role of alpha-L-fucosidase in spermatogenesis and sperm maturation.