Gait as a Quantitative Translational Outcome Measure in Angelman Syndrome

Angelman Syndrome (AS) is a genetic neurodevelopmental disorder characterized by developmental delay, lack of speech, seizures, intellectual disability, and walking and balance disorders. Recently, motor ability became an interesting outcome measure in AS, as it is broad including ataxia, hypotonia, delayed and abnormal walking and postural movements and affects nearly every individual with AS. We predict that gait presents a strong opportunity for rigorous, reliable, and quantitative metrics with direct translation to evaluate pharmacological, dietary, and genetic therapies. Numerous motoric deficits have been identified clinically. In this study, we used an innovative, automated gait analysis as well as gold standard motor behavioral assays to further delineate components of motor, coordination, balance, and gait impairments in an AS mouse model across development. Our study demonstrated marked global motoric deficits in AS mice, corroborating many previous reports. Uniquely, this is the first report of nuanced and pertinent aberrations in quantitative spatial and temporal components of gait between AS and wildtype littermate controls, that are analogous in AS individuals. These metrics were followed longitudinally to observe the progression of maladaptive gait in AS, a clinical phenotype. This has not been reported previously and contributes a substantial novel metric for therapeutic development. Taken together, these findings demonstrate the robust translational value in the study of nuanced motor outcomes, i.e., gait, for AS, as well as similar genetic syndromes, in the endeavor of therapeutic screening.

Fear Extinction and Predictive Trait-Like Inter-Individual Differences in Rats Lacking the Serotonin Transporter

Anxiety disorders are associated with a failure to sufficiently extinguish fear memories. The serotonergic system (5-hydroxytryptamine, 5-HT) with the 5-HT transporter (5-HTT, SERT) is strongly implicated in the regulation of anxiety and fear. In the present study, we examined the effects of SERT deficiency on fear extinction in a differential fear conditioning paradigm in male and female rats. Fear-related behavior displayed during acquisition, extinction, and recovery, was measured through quantification of immobility and alarm 22-kHz ultrasonic vocalizations (USV). Trait-like inter-individual differences in novelty-seeking, anxiety-related behavior, habituation learning, cognitive performance, and pain sensitivity were examined for their predictive value in forecasting fear extinction. Our results show that SERT deficiency strongly affected the emission of 22-kHz USV during differential fear conditioning. During acquisition, extinction, and recovery, SERT deficiency consistently led to a reduction in 22-kHz USV emission. While SERT deficiency did not affect immobility during acquisition, genotype differences started to emerge during extinction, and during recovery rats lacking SERT showed higher levels of immobility than wildtype littermate controls. Recovery was reflected in increased levels of immobility but not 22-kHz USV emission. Prominent sex differences were evident. Among several measures for trait-like inter-individual differences, anxiety-related behavior had the best predictive quality.

Abstract P131: Jak2 Expression In Cd11c+ Myeloid Cells Plays A Role In Salt-sensitive Hypertension Through An Enac-dependent Mechanism.

Hypertension is a major risk factor for development of cardiovascular disease. Excess dietary salt contributes to inflammation and the genesis of hypertension. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase-dependent formation of immunogenic isolevuglandin (IsoLG)-protein adducts leading to inflammation and salt-sensitive hypertension. We hypothesized that Jak2 expression, specifically in CD11c + myeloid cells, regulates expression of ENaCγ and promotes salt-sensitive hypertension. Our results indicate that deletion of Jak2 in CD11c + myeloid cells reduced the salt-induced expression of ENaCγ in CD11c+ cells. Moreover, mice lacking Jak2 in CD11c+ cells developed a blunted hypertensive response (123.8±4.7) during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride (L- NAME)/high salt model of salt-sensitive hypertension compared to their wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention (69.55±5.8) in response to high salt-feeding when compared to the wildtype litter mate controls (57.89±9.5). These results indicate that Jak2 expression in CD11c + myeloid cells plays a role in salt- sensitive hypertension through an ENaC-dependent mechanism.

Complement Properdin Determines Disease Activity in MRL/lpr Mice

Background and objects: In systemic lupus erythematosus, circulating immune complexes activate complement and, when trapped in renal capillaries, cause glomerulonephritis. Mouse models have been used in the preclinical assessment of targeting complement activation pathways to manage chronic inflammation in lupus. Properdin is the only known positive regulator of complement activation, but its role in the severity of lupus nephritis has not been studied yet. Materials and Methods: Fully characterized properdin-deficient mice were crossed with lupus prone MRL/lpr mice on C57Bl/6 background. Results: Compared to MRL/lpr properdin wildtype mice, MRL/lpr properdin-deficient mice had significantly lower anti-DNA antibody titres, TNFα and BAFF levels in serum. The qualitative glomerulonephritic score was less severe and there was significantly less serum creatinine in MRL/lpr properdin-deficient mice compared to MRL/lpr properdin wildtype littermate mice. Conclusion: Properdin plays a significant role in the severity of lupus overall and specifically in the extent of glomerulonephritis observed in MRL/lpr mice. Because MRL/lpr properdin-deficient mice had lower levels of anti-DNA antibodies, inflammatory mediators and markers of renal impairment, the study implies that properdin could constitute a novel therapy target in lupus disease.

The Role of One-Carbon Metabolism After Ischemic Stroke in an Aged Mouse Model

Objectives Nutrition is a modifiable risk factor for stroke, which is one of the leading causes of death and disability world-wide. In humans deficiencies in one-carbon metabolism, including the methyltetrahydrofolate reductase (MTHFR) polymorphism, have been linked to increased risk of stroke. The Mthfr+/− mice mouse model mimics the phenotype of the MTHFR677C – >T polymorphism. In our work using in vitro and in vivo models of ischemic stroke we have observed decreased recovery after stroke through reduced neuronal and astrocyte viability and increased apoptosis in MTHFR-deficient mice. In addition, we have previously shown dietary supplementation of one-carbon metabolites increases neuroplasticity and reduced oxidative stress after ischemic stroke. Using the MTHFR-deficient mouse model, the aim of this study was to investigate the impact of dietary supplementation with one-carbon metabolites on stroke outcome. Methods Male Mthfr+/− and wildtype littermate control mice were aged to 1.5-year-old and were placed on control diet (CD) 4-weeks prior to sensorimotor cortex damage using photothrombosis (PT), a model for ischemic stroke. Post-operatively, one group of Mthfr+/− and wildtype littermate mice were fed a supplemented diet (SD) containing 5-methylTHF, vitamin B12, and choline. Four weeks after PT damage and SD motor function was assessed and brain tissue was processed to assess lesion volume and investigate biochemical and molecular changes. Results Mthfr +/− mice fed a SD after PT did not have an impaired neuroscore compared to CD Mthfr+/− mice. When compared to CD, SD Mthfr+/− mice were able to stay on the accelerating rotarod longer and travelled further, they also used their impaired forepaw more. Total homocysteine levels in plasma and lesion volume were reduced in SD Mthfr+/+ and Mthfr+/− mice. In the brain, within the damage site, there were reduced levels of apoptotic cell death and an increased neuroprotective cellular response in SD treated Mthfr+/− mice. Conclusions This study reveals a critical role for one-carbon supplementation in supporting improvement of function after ischemic stroke. Our data suggests that in stroke affected patients, nutritional supplementation maybe an important component to post-operative care, in addition to pharmacological and rehabilitation therapies. Funding Sources NSERC.

Functional Overlap between the SEC23 Paralogs Suggests a Novel Treatment Paradigm for Congenital Dyserythropoietic Anemia Type II

Congenital dyserythropoietic anemia type II (CDAII), an autosomal recessive disease characterized by ineffective erythropoiesis and increased percentage of bi-nucleated erythroid precursors in the bone marrow (BM), results from loss of function mutations in SEC23B, which encodes a core component of COPII vesicles. Approximately 8,000 secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII vesicles, suggesting that a defect in this pathway would result in a profound systemic phenotype. However, CDAII patients exhibit a specific erythroid phenotype, with no other defects described. Mammals have 2 paralogs for SEC23, SEC23A and SEC23B. In contrast to SEC23B mutations, bi-allelic SEC23A loss of function mutations in humans result in cranio-lenticulo-sutural dysplasia, a disease characterized by skeletal defect but normal erythropoiesis. We previously demonstrated that a SEC23B-A chimeric protein composed of the first 122 amino acids of SEC23B followed by amino acids 123-765 of SEC23A overlaps in function with SEC23B, suggesting that the 2 SEC23 paralogs are functionally interchangeable. However, to rule out the possibility that the functional overlap was due to the first 122 amino acids of SEC23B, we generated a bacterial artificial chromosome (BAC) transgene that expresses the full Sec23a coding sequence from the endogenous genomic locus of Sec23b (Sec23b-a BAC). We crossed the Sec23b-a BAC to the Sec23b null allele (Sec23b-) and demonstrated that this BAC rescues the phenotype of mice deficient in Sec23b (Sec23b-/-). Therefore, we now conclusively demonstrate that the SEC23A protein functionally replaces SEC23B when expressed from the endogenous regulatory elements of Sec23b. We have previously shown that mice with erythroid-specific and pan-hematopoietic SEC23B deficiency exhibit a normal erythroid phenotype. In light of the functional overlap between SEC23A and SEC23B, we hypothesized that mice with erythroid-specific deficiency for SEC23A, alone or in combination with SEC23B, might exhibit an erythroid phenotype. First, we generated mice with erythroid-specific (EpoR-Cre) SEC23A deficiency. These mice were observed at the expected Mendelian ratios at weaning. Complete (or near complete) excision of the Sec23a floxed (Sec23afl) allele was confirmed in the erythroid cells. Peripheral blood counts, BM cellularity and morphology, and percent and distribution of BM erythroid cells among the 5 stages of maturation were indistinguishable between mice with erythroid SEC23A deficiency and wildtype littermate controls. Additionally, the percentage of bi-nucleated erythroid precursors were not increased in Sec23afl/flEpoR-Cre+ mice. Thus, mice with erythroid-specific SEC23A deficiency do not exhibit an erythroid phenotype. Similarly, mice with pan-hematopoietic SEC23A deficiency (Vav1-Cre) do not exhibit a hematologic phenotype. Next, we generated mice with Sec23a deletion and Sec23b haploinsufficiency in the erythroid compartments. These mice exhibited normal survival, a mild reduction in hemoglobin levels (p = 0.014), and a block in late erythroid maturation (Stage V erythroid cells were reduced to 22.6% compared to 30.3% in control mice; p=0.08). In contrast, mice with erythroid-specific deletion for all 4 Sec23 alleles (combined SEC23A/B deficiency) died at mid-embryogenesis exhibiting reduced size and appearing pale compared to wildtype littermate controls, with histologic evidence of dyserythropoiesis reminiscent of human CDAII. Overall, these results suggest a requirement for a threshold level of total SEC23 (combined SEC23A/B) expression in the erythroid compartment. These results also suggest that the defect in CDAII is intrinsic to the RBC. Finally, we generated K562 cells with either SEC23B or SEC23A deletion using CRISPR/Cas9 genome editing. SEC23B or SEC23A deletion alone was tolerated in the K562 cells. However, combined deletion of SEC23A and SEC23B was not tolerated. Taken together, the results summarized above demonstrate that SEC23A and SEC23B appear to compensate for one another's function in murine and human erythroid cells. This finding suggests a potential therapeutic role for increasing expression of SEC23A to compensate for SEC23B deficiency in CDAII. This work is currently ongoing. Disclosures No relevant conflicts of interest to declare.

Generation of a FLT3 Kinase Domain Point Mutation (D835Y equivalent) Knock-in Mouse Confers Less Aggressive Disease Compared with FLT3 Internal Tandem Duplication (ITD) Mice

Abstract 924 FLT3 is a tyrosine kinase receptor that is frequently mutated in acute myeloid leukemia (AML). The most common mutations involve either an internal tandem duplication (ITD) in the juxtamembrane domain or a point mutation in the tyrosine kinase domain (TKD). The most common TKD mutation is a D835Y mutation in which aspartic acid is mutated to tyrosine at residue 835. ITD mutations are reported in approximately 25% of adult AML and 15% of pediatric leukemia. TKD mutations occur in approximately 8–10% of both adult and pediatric leukemia. Both of these mutations constitutively activate FLT3, but only patients with an ITD mutation have a significantly poorer prognosis. Small molecule inhibitors against FLT3 are currently in clinical trials with some success, but resistance to single agent treatment frequently occurs. A better understanding of the molecular mechanisms behind the prognostic difference, between the two types of mutations and the activation, inhibition, and resistance of FLT3 will hopefully lead to more tailored treatments of leukemia. To help address some of these questions we have generated a knock-in mouse with an Aspartic acid to Tyrosine mutation at reside 838 (D838Y) which is homologous to the FLT3/D835Y mutation reported in the majority of human leukemia cases with a FLT3-TKD mutation. These mice have been compared to the mice with a FLT3-ITD mutation previously generated and characterized by the lab. Mice with either the FLT3/D838Y or FLT3-ITD mutation develop predominantly a myeloproliferative disease. Mice with the FLT3/D838Y mutation show a less aggressive phenotype when compared to mice with the FLT3/ITD mutation in terms of survival, spleen size, and histopathologic changes of the bone marrow, spleen and liver.8 weeks spleen wt (g)12 weeks spleen wt (g)wildtype0.079 g ± 0.011 (n=27)0.092 g ± 0.009 (n=5)D838Y/wt0.155 g ± 0.027 (n=21)0.184 g ± 0.027 (n=9)ITD/wt0.185 g ± 0.034 (n=12)0.267 g ± 0.052 (n=8) Flow cytometry analysis shows that bone marrow from 8–12 week old mice with the FLT3/D838Y mutation have a marginally expanded fraction of granulocytic/monocytic lineage cells in their bone marrow compared to their wildtype littermate controls, but a lower fraction when compared to age-matched FLT3/ITD mice. B cell development is significantly blocked at the late pro-B to pre-B transition in the FLT3/ITD mice, with very few cells developing into pre-B or more mature B cells (Li et al. Blood. 117(11): 3131-9). In contrast, FLT3/D838Y mice demonstrate an expansion of early and late pro-B cell populations, with a significant fraction of cells maturing beyond the pre-B stage to become mature B cells. In mice with the FLT3/D835Y mutation the long-term HSC (LT-HSC) compartment, responsible for the self-renewal and maintenance of the hematopoietic stem/progenitor cell pool, appears to have a greater than 2-fold expansion in the number of immunophenotypic LT-HSCs. This appears to be a functional expansion as the BM cells from FLT3/D835Y mice have long-term engraftment superiority compared with ITD and wildtype mice. The short term HSC and MPP compartments from both FLT3/D838Y and FLT3/ITD mice appear to be comparably expanded compared to wildtype controls. In human leukemia, FLT3/ITD signaling results in the activation of downstream targets, including STAT5, MAP kinase and AKT. Consistent with the intermediate phenotype observed, intracellular levels of phospho-Stat5 in the FLT3-D838Y mice were lower than levels measured in the FLT3-ITD mice, but higher than those of wildtype littermate controls. RT-PCR measured expression levels of FLT3 and Pim1 in lineage negative cells of D838Y mice fall between those of the wildtype and ITD mice. (Ratios: FLT3 – ITD/wt 3.7; D838Y/wt 2.5; Pim1 – ITD/wt 3.4; D838Y/wt 2.5). In summary, we have generated a knock-in mouse model with the FLT3 kinase domain D838Y mutation. The mice demonstrate a less aggressive phenotype when compared to knock-in mice with the FLT3/ITD mutation, analogous to the less aggressive AML seen in cases involving D835 mutations compared to ITD mutations. Both mouse models on the same mouse background provide a useful platform for understanding the molecular mechanisms of FLT3 mutations and for testing new therapeutic agents targeting FLT3. Disclosures: No relevant conflicts of interest to declare.