Jackson Laboratory Research Articles

Abstract 155: Platelet-derived Transforming Growth Factor-β1 Ameliorates AAA in a Murine Model

Background: Platelets are rapidly recruited to the forming intraluminal thrombus in abdominal aortic aneurysm (AAA), stabilizing the aortic wall and preventing rupture. Adhesion and activation of platelets releases a mix of inflammatory mediators such as transforming growth factor β (TGFβ), which mediates cellular proliferation, inflammatory signaling, and fibrosis. Importantly, while TGFβ neutralization results in increased rupture and disease progression in mouse models of AAA, the source of protective TGFβ signaling remains controversial. We hypothesized that platelet-derived TGFβ1 attenuates aortic growth in the elastase AAA model. Methods and Results: Ldlr -/- mice with platelet-specific deletion of Tgfβ1 were produced by breeding hemizygous male mice expressing Cre under the control of the Pf4 promoter to female Tgfβ1 floxed mice (Jackson Labs). Mice were subjected to laparotomy and topical elastase application to the abdominal aorta (10 mg/mL porcine pancreatic elastase for 5 minutes) and sacrificed at day 28 to determine AAA progression via aortic diameter. Male TGFβ1-Pf4 Cre+ mice (n=8) had significantly increased abdominal aortic diameters compared to Male TGFβ1-Pf4 Cre- mice (n=7) ( Cre- : 1.70 ± 0.12 mm; Cre+ : 2.66 ± 0.25 mm; P < 0.004). Additionally, 25% of male TGFβ1-Pf4 Cre+ mice died of AAA rupture compared to 0% in the TGFβ1-Pf4 Cre- cohort. To determine if these actions were occurring via TGFβ1 activation of the TGFβ receptor 2 (βr 2 ), Ldlr -/- /βr2 Flox /PF4 Cre+ and Cre- mice were created, similarly to the previously described Tgfβ1 mice. Male βr2-Pf4 Cre+ (n=7) had increased aortic diameter compared to βr2-Pf4 Cre- (n=6) ( Cre- : 1.70± 0.20 mm; Cre+ : 2.45 ± 0.38 mm; P < 0.138). Additionally, female βr2-Pf4 Cre+ (n=9) also had significantly larger aortic diameters compared to βr2-Pf4 Cre- (n = 5) ( Cre- : 1.27± 0.04 mm; Cre+ : 1.90 ± 0.23 mm; P < 0.01). Conclusions: Our results further support TGFβ signaling playing a protective role in AAA. More specifically, platelet-derived TGFβ1 ligand and signaling via platelet TGFβr2 mitigates aortic diameter expansion. Further research into the pertinent TGFβ isoforms and the mechanism of TGFβr2 activation will ideally yield insights into therapeutic targets for an, as yet, intractable disease.

Microenvironment regulates antibody secretion by IgA+ plasma cells.

Abstract IgA secreting plasma cells (PCs) provide a critical supply of antibodies for barrier functions against pathogens. These cells express unique tissue-driven transcriptional profiles in their respective niches in the bone marrow (BM) and gut. We previously demonstrated that both tissues sustain long-lived IgA+ PCs, but the cell intrinsic and extrinsic signals responsible for survival and function in the gut remain largely unknown. Understanding how the microenvironment regulates tissue resident IgA+ PC functions has implications for maintaining intestinal homeostasis and protection from disease. We found that BM IgA+ PCs functionally differ from gut IgA+ PCs as they secrete significantly more antibody. To assess if this phenotype could be due to metabolic consequences, we profiled IgA+ PCs in both tissues. BM and gut IgA+ PCs have similar glucose and fatty acid uptake. However, BM IgA+ PCs have increased mitochondrial dependence, while gut IgA+ PCs have increased glycolytic capacity. We next altered the composition of the microbiome to determine the role of gut microbes in regulating tissue resident PCs. Our initial experiments used C57BL/6J mice with a well-defined low-complexity microbiome (from Jackson Labs). Co-housing Jax mice with animals containing a more diverse set of microbes led to an increase in antibody secretion. Herein, we identified that the tissue microenvironment and gut microbiome influences PC function. We aim to dissect the mechanisms involved in microbiome driven changes in PC phenotype. Ultimately, understanding how the microenvironment regulates intensity of PC antibody secretion provides a tool in targeting induction of systemic and mucosal responses and will improve vaccine design and development. Supported by grants from NIH (R35 GM147560, R03 AI156481)

Meeting report of the annual workshop on Principles and Techniques for Improving Preclinical to Clinical Translation in Alzheimer's Disease research.

The second annual 5-day workshop on Principles and Techniques for Improving Preclinical to Clinical Translation in Alzheimer's Disease Research was held October 7-11, 2019, at The Jackson Laboratory in Bar Harbor, Maine, USA, and included didactic lectures and hands-on training. Participants represented a broad range of research across the Alzheimer's disease (AD) field, and varied in career stages from trainees and early stage investigators to established faculty, with attendance from the United States, Europe, and Asia. In line with the National Institutes of Health (NIH) initiative on rigor and reproducibility, the workshop aimed to address training gaps in preclinical drug screening by providing participants with the skills and knowledge required to perform pharmacokinetic, pharmacodynamics, and preclinical efficacy experiments. This innovative and comprehensive workshop provided training in fundamental skill sets for executing in vivo preclinical translational studies. The success of this workship is expected to translate into practical skills that will enablethe goals of improving preclinical to clinical translational studies for AD. Nearly all preclinical studies in animal models have failed to translate to successful efficacious medicines for Alzheimer's disease (AD) patients. While a wide variety of potential causes of these failures have been proposed,deficiencies in knowledge and best practices for translational research are not being sufficiently addressed by common training practices. Here we present proceedings from an annual NIA-sponsored workshop focused specifically on preclinical testing paradigms for AD translational research in animal models aimed at enabling improved preclinical to clinical translation for AD.

Understanding Patterns of Life History Trait Covariation in an Untapped Resource, the Lab Mouse.

AbstractThrough artificial selection and inbreeding, strains of laboratory mice have been developed that vary in the expression of a single or suite of desired traits valuable to biomedical research. In addition to the selected trait(s), these strains also display variation in pelage color, body size, physiology, and life history. This article exploits the broad phenotypic variation across lab mouse strains to evaluate the relationships between life history and metabolism. Life history variation tends to exist along a fast-slow continuum. There has been considerable interest in understanding the ecological and evolutionary factors underlying life history variation and the physiological and metabolic processes that support them. Yet it remains unclear how these key traits scale across hierarchical levels, as ambiguous empirical support has been garnered at the intraspecific level. Within-species investigations have been thwarted by methodological constraints and environmental factors that obscure the genetic architecture underlying the hypothesized functional integration of life history and metabolic traits. In this analysis, we used the publicly available Mouse Phenome Database by the Jackson Laboratory to investigate the relationships among life history traits (e.g., body size, reproduction, and life span) and metabolic traits (e.g., daily energy expenditure and insulin-like growth factor 1 concentration). Our findings revealed significant variation in reproductive characteristics across strains of mice as well as relationships among life history and metabolic traits. We found evidence of variation along the fast-slow life history continuum, though the direction of some relationships among these traits deviated from interspecific predictions laid out in previous literature. Furthermore, our results suggest that the strength of these relationships are strongest earlier in life.

Abstract 1: Mouse models of cancer available from The Jackson Laboratory

Abstract Genetically defined mice are a cornerstone of cancer research. The Jackson Laboratory (JAX) offers more than 13,500 unique mouse lines to the scientific community, many useful in advancing cancer research. Available are mouse models for specific cancers, xenograft recipients, immunodeficient platforms for PDX studies and multi-purpose tool strains having CRISPR/Cas9 expression, conditional/inducible expression (Cre-lox, FLP-frt, Tet-On/-Off), optogenetic function and calcium-sensing technologies. This poster will highlight some recently added models, more than 150 of which have cancer research applications. The portfolio of strains useful for transplantation/engraftment and “humanized” mouse research continues to expand and complement the NSG (NOD scid IL2rγc-/-) model as ideal tools for engraftment with a wide range of malignant or non-malignant human or mouse tissues. Fluorescent protein-expressing strains include the Traffic Light Reporter models for tracking of DNA repair pathways and the Gene Editing Reporter models for detection of homology directed repair (HDR) or non-homologous end joining (NHEJ) repair events. As a tool for gene targeting, the RosaBxb-GT/GA mice are designed to allow insertion of ~40kb of desired DNA sequence into the Gt(ROSA)26Sor locus. In addition to safeguarding each strain by cryopreservation, JAX has a comprehensive genetic quality control program that confirms mutation identity and genetic background, and screens for unwanted alleles (such as stray GFP, Cre, neo, etc.). Search the comprehensive collection of innovative mouse models using our improved JAX Mouse Search site (mice.jax.org). This resource includes models created by many generous donating institutions. Researchers are encouraged to donate their mouse lines via a very short Strain Submission form (jax.org/donate-a-mouse). Please also visit the JAX Oncology webpage for cancer research related strains and resources (jax.org/jax-mice-and-services/solutions-by-therapeutic-area/oncology). JAX is supported by the NIH, The Howard Hughes Medical Institute and other private charitable foundations. Citation Format: Jason Beckwith, Jen Merriam, Alicia Valenzuela, Darcy Pomerleau, Crystal Davis. Mouse models of cancer available from The Jackson Laboratory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1.

Abstract 2347: Producing genetically defined CAFs for the study of hypoxia signaling in the pancreatic cancer TME

Abstract Introduction (or Objective): Pancreatic ductal adenocarcinoma (PDAC) is characterized by having an extensive desmoplastic stroma that contributes to its resistance to therapeutic treatments resulting in a poor prognosis. Cancer associated fibroblasts (CAFs) are the most prevalent stromal cells within the pancreatic tumor microenvironment (TME), making up roughly 70-80% of the tumor volume. Pancreatic tumors are extremely hypoxic due to this dense stromal infiltration. We previously showed that CAFs in PDAC regulate macrophage activity and polarization in a HIF2-dependent manner, revealing the importance of hypoxia signaling in stromal cells. PDAC-CAFs also participate in cross talk with cancer cells inducing cancer malignancy, angiogenesis, metastasis, and drug resistance. In this study, we utilized a novel mouse model to produce genetically defined CAFs to study how HIFs (including HIF1a, HIF2a, and their binding partner ARNT) contribute to the function of CAFs in the pancreatic cancer TME. Methods: LSL-tdTomato mice were obtained from Jackson laboratories. TdTomato mice were bred with aSMACreERT2/+ mice, and their progeny were bred with Hif1afl/fl, Hif2afl/fl and ARNTfl/fl mice to produce LSL-tdTomato; aSMACreERT2/+; HIF1afl/fl (HIF1-KO)/HIF2afl/fl (HIF2-KO)/ARNTfl/fl (ARNT-KO) mice, respectively. KPC tumor cells (KrasG12D;Trp53R172H) were then injected orthotopically into the pancreas of these mice. After one month, tumors were harvested and digested to allow for CAF isolation. Digested single cells were then sorted for tdTomato expression to specifically isolate aSMA+ CAFs. All mice are on a C57BL/6J genetic background. Results: We isolated genetically defined CAFs lacking either HIF1, HIF2 and ARNT in a syngeneic orthotopic tumor model. After dissociating solid tumors and isolating tdTomato+ cells, we isolated early passage CAFs as verified by expression of fibroblast markers (ACTA1, COL1A1, FN1, etc.) without epithelial markers (EPCAM, KRT19). Genetic loss of HIF1, HIF2 or ARNT was verified via western blot and qPCR analysis. Conclusion: We created a Cre-driven model to specifically label CAFs and genetically manipulate hypoxia signaling that can be used with any desired promoter. This model allows the isolation and characterization of spatially and temporally genetically defined stromal populations, which may enable downstream syngeneic transplantation. We believe that this powerful system could be used to interrogate each stromal component in a definitive fashion without the need for more complex multiallelic mouse models. Citation Format: Sahar B. Fattani, Matthew T. Cribb, Abagail M. Delahoussaye, Nefetiti P. Mims, Natividad R. Fuentes, Cullen M. Taniguchi. Producing genetically defined CAFs for the study of hypoxia signaling in the pancreatic cancer TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2347.

Abstract 4244: Black soybean seed coat extract suppress intestinal tumorigenesis in APCmin mice

Abstract Background: Colorectal cancer (CRC) is a major cause of cancer-related deaths in Japan and worldwide. Since CRC has been reported to have close relationship with diet, it seems significant to identify dietary constituents that might suppress occurrence of CRC. Recently, an epidemiological investigation for cancer incidence in Japan revealed that consumptions of black soybeans seem to reduce incidence of several cancers, especially colorectal cancer. Black soybeans coat extract (BE) is known to contain rich procyanidins which have many beneficial physiological activities such as anticancer effects. Therefore, we hypothesized that BE may have inhibitory effects to intestinal tumorigenesis and investigated using APCmin/+ mice models. Materials and Methods: APCmin/+ male mice in the C57BL/6 genetic background were obtained from Jackson Laboratories. APCmin/+ genotypes of each mouse were confirmed by polymerase chain reaction analysis (PCR) using DNA extracted from the tip of mouse tails. Mice were weighed weekly once and monitored regularly for any signs of weight loss. APC min/+ mice at 4 weeks of age were randomly divided into three groups with 5 mice, and fed by either AIN-76A diet, AIN-76A diet containing 0.05% or 0.05% BE for 8 weeks. At 12 weeks of age, intestines were removed from each mouse and then sliced longitudinally. The small intestine was cut into three equally parts: proximal, middle, and distal. Numbers and sizes of intestinal polyps in each portion were evaluated under Video-Zoom-microscope XV-440. Intestinal polyps were categorized by size into 1-2 mm, 2-3 mm and >3 mm. Other parts of the intestines were placed in 10% phosphate-buffered formalin for histopathology and immunohistochemical (PCNA, β-catenin, and MUC2) analyses. Results: Numbers of polyps in mice fed with containing 0.05% or 0.5% BE diet were significantly smaller than in mice fed without BE (P < 0.05). As for size of polyps, the number of polyps of 1 to 3 mm in size was decreased in mice with BE, however, there were no significant difference for numbers of polyps more than 3 mm in size regarding diets with or without BE. With 0.05% BE diet, reduction rates for occurrence of polyps were 81% for proximal, 48% for middle, and 47% for distal positions, while in with 0.5% BE diet, those reduction rates were 71% for proximal, 59% for middle, and 58% for distal positions, respectively. From these results, the BE, either 0.05% or 0.05% concentration, showed inhibitory effects for tumorigeneses in the small intestine of APC min/+ mice. In histological analyses, the BE was induced higher expression of MUC2-positive cells in normal intestinal lesions and lower expression of β-catenin in tumor sites Conclusions: The BE showed inhibitory effects in occurrence and growth of intestinal polyps in APCmin/+ mice models. With further evaluation of mechanisms, the BE could be a promising nutrition for prevention of CRC in human. Citation Format: Yasuyuki Shimizu, Shunta Hirano, Yoko Yamashita, Tsuyoshi Fukumoto, Naritoshi Mukumoto, Ai Nakaoka, Namiko Hoshi, Takeaki Ishihara, Daisuke Miyawaki, Ashida Hitoshi, Ryohei Sasaki. Black soybean seed coat extract suppress intestinal tumorigenesis in APCmin mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4244.

Renal Function in Aged C57BL/6J Mice Is Impaired by Deposition of Age-Related Apolipoprotein A-II Amyloid Independent of Kidney Aging

Spontaneous and age-related amyloidosis has been reported in C57BL/6J mice. However, the biochemical characteristics of age-related amyloidosis remain unclear. Herein, the age-related prevalence of amyloidosis, the types of amyloid fibril proteins, and the effects of amyloid deposition were investigated in renal function in C57BL/6J mice. The results obtained revealed a high incidence of amyloidosis in C57BL/6J mice originating from The Jackson Laboratory as well as the deposition of large amounts of amyloid in the glomeruli of aged mice. The amyloid fibril protein was identified as wild-type apolipoprotein A-II (ApoA-II). Induction of amyloid deposition in 40-week-old mice, equivalent to that of spontaneous development in 80-week-old mice, to rule out the effects of aging, revealed subsequent damage to kidney function by amyloid deposits. Furthermore, amyloid deposition in the mesangial region decreased podocyte density, compromised foot processes, and led to the accumulation of fibroblast growth factor 2 in glomeruli. Collectively, these results suggest that ApoA-II deposition is a general pathology in aged C57BL/6J mice and is dependent on supplier colonies. Therefore, the effects of age-related amyloid deposition need to be considered in research on aging in mice.

Correction: Temporal and spatially controlled APP transgene expression using Cre-dependent alleles

There were errors in Disease Models & Mechanisms (2022) 15, dmm049330 (doi:10.1242/dmm.049330).The authors recently discovered that one of the Cre-dependent transgenic mouse lines used in the study is not correctly described within the article. The mouse line labelled as R26-APP in the paper was originally described as containing both the Swedish and Iberian mutations, but carries only the Iberian mutation. This was discovered by The Jackson Laboratory when they sequenced through the complete insert to confirm the allele before entering the line into their repository for distribution. The authors also discovered an unintended point mutation located just outside the Aβ domain, M647T by APP695 numbering (M722T by APP770 numbering). They propose that the model should be identified as R26-APPIbe rather than as R26-APP.A diagram of the allele that appears in Fig. 2A has been corrected:In addition, a section describing the cloning methods in the Materials and Methods has been updated. The corrected and original versions are shown below:‘Cloning of R26-LSL-hAPPIbe transgene’ section (corrected). Human APPIbe 695 amino acid cDNA was generated in two steps. The N-terminal 1592 nucleotides of huAPP695 were cut from pcDNA3.1-sAPPα (a gift from Hui Zheng, BCM) with HindIII and SacI and subcloned into the same sites of transfer plasmid L4440 (Addgene, #1654). The remaining fragments were amplified from a human cDNA library using a two-step PCR method. Both PCR products contained the Ibe mutation as well as an overlap region needed to serve as an extended template for the second PCR. The first C-terminal fragment was amplified using hAPP C-terminus forward primer 5′-AACGAAAACCACCGTGGAGCT-3′ and Ibe reverse primer 5′-AAGGTGATGACGAACACTGTCG-3′. The second C-terminal fragment was amplified by Ibe forward primer 5′-CGACAGTGTTCGTCATCACCTT-3′ and hAPP C-terminus reverse primer 5′-GCGCGGATATCCTAGTTCTGCATCTGCTCAAAG-3′. The complete hAPP695Ibe C-terminus was then amplified from a mixture of the two shorter products using hAPP CT forward primer and hAPP CT reverse primer. The purified huAPPIbe C-terminal PCR fragment was digested with EcoRV and SacI and ligated into the EcoRV/SacI sites of L4440-APP(1-1592), resulting in L4440-APPIbe. The complete huAPPIbe sequence was transferred into an intermediary expression plasmid, which was digested with MluI and FseI and cloned into an Ai3-derived ROSA26 targeting vector (Madisen et al., 2010) to generate the final transgene construct pJJ-hAPPIbe. The modified Ai3 targeting vector retained the original Ai3 Cre-dependent CAG-loxP-stop-loxP sequence but removed the PGK-DTA negative selection cassette and shortened the 3′ targeting arm to reduce the overall vector size. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).‘Cloning of R26-LSL-hAPPSwe/Ibe transgene’ section (original). Human APPSwe/Ibe 695 amino acid cDNA was amplified from the pBR322-APPSwe/Ind plasmid used to generate the Tg CRND8 mouse model (Chishti et al., 2001) (a gift from David Westaway, University of Alberta, Canada) with primers that converted the original Indiana mutation V717F into the Iberian mutation I716F (point mutation numbering is for the standard human APP 770 amino acid isoform). The N-terminus of APPSwe/Ibe 695 was amplified using the forward primer 5′-GGCCGGCCGCCACCATGCTGCCCGGTTTGGCACT-3′ and reverse primer 5′-AAGGTGATGACGAACACTGTCG-3′; the C-terminus was amplified using the forward primer 5′-CGACAGTGTTCGTCATCACCTT-3′ and reverse primer 5′-GCGCGACGCGTCTAGTTCTGCATCTGCTCAAAGAA-3′. The resulting hAPPSwe/Ibe fragments were combined by amplification using the forward primer 5′-GGCCGGCCGCCACCATGCTGCCCGGTTTGGCACT-3′ and reverse primer 5′-GCGCGACGCGTCTAGTTCTGCATCTGCTCAAAGAA-3′. The complete hAPPSwe/Ibe 695 cassette was digested with MluI and FseI, and cloned into an Ai3-derived ROSA26 targeting vector (Madisen et al., 2010) that had been cut with the same enzymes to generate the final transgene construct pJJ-hAPPSwe/Ibe. The modified Ai3 targeting vector retained the original Cre-dependent CAGloxP-stop-loxP sequence but removed the PGK-DTA negative selection cassette and shortened the 3′ targeting arm to reduce the overall vector size. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).Throughout the paper, where the text refers to R26-APP, R26-APPSwe/Ibe or ROSA26-CAG-LSL-APPSwe/Ibe, corrections have been made to R26-APPIbe or ROSA26-CAG-LSL-APPIbe. The model will still be distributed by The Jackson Laboratory as stock #037319, but using the line name R26-APPIbe. The study results and conclusions remain unaffected.Both the online full-text and PDF versions have been updated. The authors apologise to readers for these errors and any inconvenience they may have caused.

A new FRDA mouse model [Fxn null:YG8s(GAA) > 800] with more than 800 GAA repeats.

Friedreich's ataxia (FRDA) is an inherited recessive neurodegenerative disorder caused by a homozygous guanine-adenine-adenine (GAA) repeat expansion within intron 1 of the FXN gene, which encodes the essential mitochondrial protein frataxin. There is still no effective therapy for FRDA, therefore the development of optimal cell and animal models of the disease is one of the priorities for preclinical therapeutic testing. We obtained the latest FRDA humanized mouse model that was generated on the basis of our previous YG8sR, by Jackson laboratory [YG8JR, Fxn null:YG8s(GAA) > 800]. We characterized the behavioral, cellular, molecular and epigenetics properties of the YG8JR model, which has the largest GAA repeat sizes compared to all the current FRDA mouse models. We found statistically significant behavioral deficits, together with reduced levels of frataxin mRNA and protein, and aconitase activity in YG8JR mice compared with control Y47JR mice. YG8JR mice exhibit intergenerational GAA repeat instability by the analysis of parent and offspring tissue samples. Somatic GAA repeat instability was also detected in individual brain and cerebellum tissue samples. In addition, increased DNA methylation of CpG U13 was identified in FXN GAA repeat region in the brain, cerebellum, and heart tissues. Furthermore, we show decreased histone H3K9 acetylation and increased H3K9 methylation of YG8JR cerebellum tissues within the FXN gene, upstream and downstream of the GAA repeat region compared to Y47JR controls. These studies provide a detailed characterization of the GAA repeat expansion-based YG8JR transgenic mouse models that will help investigations of FRDA disease mechanisms and therapy.

A mouse model of cone photoreceptor function loss (cpfl9) with degeneration due to a mutation in Gucy2e.

During routine screening of mouse strains and stocks by the Eye Mutant Resource at The Jackson Laboratory for genetic mouse models of human ocular disorders, we identified cpfl9, a mouse model with cone photoreceptor function loss. The mice exhibited an early-onset phenotype that was easily recognized by the absence of a cone-mediated b-wave electroretinography response and by a reduction in rod-mediated photoresponses at four weeks of age. By genetic mapping and high-throughput sequencing of a whole exome capture library of cpfl9, a homozygous 25 bp deletion within exon 11 of the Gucy2e gene was identified, which is predicted to result in a frame shift leading to premature termination. The corresponding protein in human, retinal guanylate cyclase 1 (GUCY2D), plays an important role in rod and cone photoreceptor cell function. Loss-of-function mutations in human GUCY2D cause LCA1, one of the most common forms of Leber congenital amaurosis, which results in blindness at birth or in early childhood. The early loss of cone and reduced rod photoreceptor cell function in the cpfl9 mutant is accompanied by a later, progressive loss of cone and rod photoreceptor cells, which may be relevant to understanding disease pathology in a subset of LCA1 patients and in individuals with cone-rod dystrophy caused by recessive GUCY2D variants. cpfl9 mice will be useful for studying the role of Gucy2e in the retina.

PS-BPB10-2: MOLECULAR INSIGHT INTO SALT-SENSITIVITY; THE ROLE OF ATF3 AND THE GENERATION OF TUBULAR SPECIFIC KO MICE. (1)

Objectives: Current research including ours suggested pivotal roles of expressions of and regulations of tubular ion transporters for developments of sodium sensitivity and hypertension. Orlov et al. demonstrated using rat cardiomyocytes three different genes which are activated according to the elevation of intra-cellular Na ion concentration. (2015) To elucidate detailed biological roles of these genes in tubules, we performed current experiments. Methods: We tested expressions of three genes for cDNA from kidneys of Nedd4 2 C2 KO mice using quantitative RT PCR methods. Subsequently, we performed crossbreeding experiments between floxed target gene mice (generous gift from Prof. Kitamura, Tokyo Medical and Dental University) and Ksp-cre mice (Jackson Laboratory), which cre-recombinase expresses specifically along urinary tubules to generate mice with cre+, flox/flox genotype. Results: We found expressions of Atf3 are significantly changed due to oral sodium intake and diuretics treatment in Nedd4–2 C2 KO mice. We successfully generated mice with cre+, Atf3 flox/flox mice for current experiments. Under normal salt diet, we could not find any significant differences between cre + and minus mice in terms of systolic blood pressure, water intake, urine volume, urinary sodium excretions, urinary pH, and urinary osmotic pressures using metabolic cage experiments. We found, under high salt intake, gene expressions of both classic renin and tubular alternative renin are significantly suppressed for 30 to 70% in KO mice by quantitative RT PCR and immunohistochemistry imaging. Conclusions: Tubular epithelial Atf3 genes are involved in regulations of renin gene expressions in accordance with intracellular Na ion concentrations. Therefore, there are possibilities that tubular specific Atf3 KO mice shows blood pressure change under various salt intake and diuretics treatment.

Generalisable long COVID subtypes: findings from the NIH N3C and RECOVER programmes.

Stratification of patients with post-acute sequelae of SARS-CoV-2 infection (PASC, or long COVID) would allow precision clinical management strategies. However, long COVID is incompletely understood and characterised by a wide range of manifestations that are difficult to analyse computationally. Additionally, the generalisability of machine learning classification of COVID-19 clinical outcomes has rarely been tested. We present a method for computationally modelling PASC phenotype data based on electronic healthcare records (EHRs) and for assessing pairwise phenotypic similarity between patients using semantic similarity. Our approach defines a nonlinear similarity function that maps from a feature space of phenotypic abnormalities to a matrix of pairwise patient similarity that can be clustered using unsupervised machine learning. We found six clusters of PASC patients, each with distinct profiles of phenotypic abnormalities, including clusters with distinct pulmonary, neuropsychiatric, and cardiovascular abnormalities, and a cluster associated with broad, severe manifestations and increased mortality. There was significant association of cluster membership with a range of pre-existing conditions and measures of severity during acute COVID-19. We assigned new patients from other healthcare centres to clusters by maximum semantic similarity to the original patients, and showed that the clusters were generalisable across different hospital systems. The increased mortality rate originally identified in one cluster was consistently observed in patients assigned to that cluster in other hospital systems. Semantic phenotypic clustering provides a foundation for assigning patients to stratified subgroups for natural history or therapy studies on PASC. NIH (TR002306/OT2HL161847-01/OD011883/HG010860), U.S.D.O.E. (DE-AC02-05CH11231), Donald A. Roux Family Fund at Jackson Laboratory, Marsico Family at CU Anschutz.

Effect of Husbandry Practices on the Fecal Microbiota of C57BL/6J Breeding Colonies Housed in 2 Different Barrier Facilities in the Same Institution.

Evidence showing a relationship between the mouse gut microbiome and properties such as phenotype and reaction to therapeutic agents and other treatments has increased significantly over the past 20 to 30 y. Recent concerns regarding the reproducibility of animal experiments have underscored the importance of understanding this relationship and how differences in husbandry practices can affect the gut microbiome. The current study focuses on effects of different barrier practices in 2 barrier facilities at the same institution on the fecal microbiome of breeding C57Bl/6J mice. Ten female and 10 male C57Bl/6J mice were obtained in one shipment from Jackson Laboratories and were housed under different barrier conditions upon arrival. Fecal samples were collected on arrival and periodically thereafter and were sent to TransnetYX for microbiome analysis. Mice used for collection of feces were housed as breeding pairs, with a total of 5 breeding pairs per barrier. An additional fecal sample was collected from these mice at 8 wk after arrival. One F1 female and one F1 male from each breeding cage were housed as brother-sister breeding pairs and a fecal sample was collected from them at 8 wk of age. Brother-sister breeding colonies were continued through F3, with fecal samples for microbiome analysis were collected from each generation at 8 wk of age. Breeding colonies in the 2 barriers showed differences in relative abundance, α -diversity, and β -diversity. Our data indicate that differences in barrier husbandry practices, including the use of autoclaved cages, the degree of restricted access, feed treatment practices, and water provision practices, can affect fecal microbiome divergence in both the parental and filial generations of different breeding colonies. To our knowledge, this is the first study to examine the effect of barrier husbandry practices on the microbiome of breeding colonies through the F3 generation.

Phenotyping of lenses from eyes of P301S tau mice

AbstractBackgroundWe previously discovered that Aβ accumulates in the supranuclear region of the lens in the eyes of people with Alzheimer’s Disease (AD) (Goldstein et al., 2003) and Down Syndrome (DS; (Moncaster et al., 2010) and also in the Tg2576 APP swedish mutation AD mouse model (Moncaster et al., 2022). Aβ interacts with crystallin proteins in the lens to create light scattering aggregates that eventually manifest as cortical cataracts. Tau has previously been reported to be expressed in the lens of the eye (Bai et al., 2007; Zhao et al., 2013). Here we investigated whether cataracts may develop in the P301S tau model.MethodP301S mice were bred and maintained at Boston University School of Medicine. Breeder mice were purchased from Jackson Laboratories, Bar Harbor, ME. Male and Female transgenic and non‐transgenic mice (n = 5‐6 per group) were sacrificed at ages 12‐13 months. This timepoint was chosen as it is the limit of lifespan for P301S transgenic mice. Mice were perfused with phosphate buffer saline, lenses were isolated and then imaged under two different sources of light using a D70 digital Nikon camera and a custom‐adapted Zeiss stereophotomicroscope.ResultThere was no overt difference in lens phenotype between the P301S transgenic and non‐transgenic mice. No cataracts were observed in these lenses at 12‐13 months of age.ConclusionThere was no overt difference in lens phenotype between the P301S transgenic and non‐transgenic mice. Based on our current and previous results, the data suggests that Aβ may play a more significant role in lens pathology in AD than tau.

Adaptive Sentinel Testing in Workplace for COVID-19 Pandemic.

Testing and isolation of infectious employees is one of the critical strategies to make the workplace safe during the pandemic for many organizations. Adaptive testing frequency reduces cost while keeping the pandemic under control at the workplace. However, most models aimed at estimating test frequencies were structured for municipalities or large organizations such as university campuses of highly mobile individuals. By contrast, the workplace exhibits distinct characteristics: employee positivity rate may be different from the local community because of rigorous protective measures at workplace, or self-selection of co-workers with common behavioral tendencies for adherence to pandemic mitigation guidelines. Moreover, dual exposure to COVID-19 occurs at work and home that complicates transmission modeling, as does transmission tracing at the workplace. Hence, we developed bi-modal SEIR (Susceptible, Exposed, Infectious, and Removed) model and R-shiny tool that accounts for these differentiating factors to adaptively estimate the testing frequency for workplace. Our tool uses easily measurable parameters: community incidence rate, risks of acquiring infection from community and workplace, workforce size, and sensitivity of testing. Our model is best suited for moderate-sized organizations with low internal transmission rates, no-outward facing employees whose position demands frequent in-person interactions with the public, and low to medium population positivity rates. Simulations revealed that employee behavior in adherence to protective measures at work and in their community, and the onsite workforce size have large effects on testing frequency. Reducing workplace transmission rate through workplace mitigation protocols and higher sensitivity of the test deployed, although to a lesser extent. Furthermore, our simulations showed that sentinel testing leads to only marginal increase in the number of infections even for high community incidence rates, suggesting that this may be a cost-effective approach in future pandemics. We used our model to accurately guide testing regimen for three campuses of the Jackson Laboratory.

Effects of an exogenous ketone ester using multi-omics in skeletal muscle of aging C57BL/6J male mice.

Exogenous ketone ester supplementation provides a means to increase circulating ketone concentrations without the dietary challenges imposed by ketogenic diets. Our group has shown that oral R,S-1,3, butanediol diacetoacetate (BD-AcAc2) consumption results in body weight loss or maintenance with moderate increases in circulating ketones. We have previously shown a diet consisting of 25% BD-AcAc2 can maintain lean body mass (LBM) and induce fat mass (FM) loss in young, healthy male mice, but the underlying mechanisms are still unknown. Therefore, the purpose of this study was to determine if a diet consisting of 25% BD-AcAc2 (ketone ester, KE) would alter body composition, transcriptional regulation, the proteome, and the lipidome of skeletal muscle in aged mice. We hypothesized that the KE group would remain weight stable with improvements in body composition compared to controls, resulting in a healthy aging phenotype. Male C57BL/6J mice (n = 16) were purchased from Jackson Laboratories at 72 weeks of age. After 1 week of acclimation, mice were weighed and randomly assigned to one of two groups (n = 8 per group): control (CON) or KE. A significant group by time interaction was observed for body weight (P < 0.001), with KE fed mice weighing significantly less than CON. FM increased over time in the control group but was unchanged in the KE group. Furthermore, LBM was not different between CON and KE mice despite KE mice weighing less than CON mice. Transcriptional analysis of skeletal muscle identified 6 genes that were significantly higher and 21 genes that were significantly lower in the KE group compared to CON. Lipidomic analysis of skeletal muscle identified no differences between groups for any lipid species, except for fatty acyl chains in triacylglycerol which was 46% lower in the KE group. Proteomics analysis identified 44 proteins that were different between groups, of which 11 were lower and 33 were higher in the KE group compared to CON. In conclusion, 72-week-old male mice consuming the exogenous KE, BD-AcAc2, had lower age-related gains in body weight and FM compared to CON mice. Furthermore, transcriptional and proteomics data suggest a signature in skeletal muscle of KE-treated mice consistent with markers of improved skeletal muscle regeneration, improved electron transport chain utilization, and increased insulin sensitivity.

Inhibition of Hypoxia Inducible Factor Prolyl Hydroxylase By FG-4592 (Roxadustat) Increases Erythropoietic Stimulation in a Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is a devastating monogenic disorder caused by a mutation in the β-globin gene, leading to production of mutant hemoglobin S (HbS). Abnormal polymerization of deoxygenated HbS causes sickling of red blood cells (RBCs), resulting in painful vaso-occlusive crises and chronic hemolytic anemia. Etiologies of chronic anemia in SCD are multifactorial, including intravascular and extravascular hemolysis, inadequate erythropoietin (EPO) production, and abnormal iron homeostasis (Xu and Thein, Blood 2022). Hydroxyurea (HU) is the standard-of-care drug therapy for SCD and primarily reduces HbS polymerization through induction of fetal Hb (HbF) expression. However, it only modestly improves anemia and does not address other causes of anemia in SCD. Additionally, lower Hb levels in SCD are associated with chronic kidney disease (CKD), stroke, and pulmonary hypertension, arguing for the need for additional therapies targeting anemia in SCD. Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a class of drugs developed for treatment of anemia in CKD and end-stage renal disease. HIF-PHIs stimulate erythropoiesis by inhibiting prolyl hydroxylase and von Hippel-Lindau (VHL)-mediated degradation of HIF-α, leading to increased hypoxic signaling and promotion of HIF-induced EPO expression in the kidneys and liver. Stimulation of erythropoiesis also increases production of erythroferrone, the main regulator of hepcidin, and suppression of hepcidin levels by HIF-PHIs may improve iron mobilization. Furthermore, HIF-PHIs can induce HbF expression in erythroblasts derived from CD34+ hematopoietic stem and progenitor cells (HSPCs) (Hsieh et al.,Blood 2007, Feng et al., ASH abstract 2021), suggesting multiple potential therapeutic benefits in SCD. Therefore, we characterized the effect of the HIF-PHI FG-4592 (roxadustat), on HbF induction in CD34+ HSPCs and on hematologic markers, EPO and hepcidin expression in a mouse model of SCD. CD34+ HSPCs from 3 donors were differentiated in vitro to erythroblasts with vehicle, 20 μM of FG-4592, or 50 μM of HU. Flow cytometric analysis on Day 9 showed that FG-4592 treatment increased % F-cells by 1.47-fold (±0.45), compared to a 2.21-fold (±0.89) increase with HU (Fig. 1). We then transplanted lethally irradiated (1000 rads) male 6-8-week-old C57BL/6J mice with bone marrow from transgenic sickle cell mice (Hb SS; Townes model, Jackson Laboratory). Twelve weeks after transplantation, mice with >80% engraftment of the donor HbSS phenotype, determined by Hb electrophoresis, were used for subsequent experiments. Vehicle, 25 mg/kg FG-4592, or 50 mg/kg FG-4592 was administered three times weekly through intraperitoneal injection for 8 weeks. All mice underwent retro-orbital bleeding at baseline (48 h pretreatment), 2, 4, 6, and 8 weeks of treatment for hematological evaluation, including Hb, hematocrit, and reticulocyte % (Fig. 1). At the end of 8 weeks, livers, kidneys, and plasma were collected. Hb and hematocrit levels increased in the 50 mg/kg FG-4592 group compared to the other groups at 4 weeks but decreased by 8 weeks for all groups. Reticulocyte % also decreased in all groups at the 4- and 8-week timepoints. Red cell distribution width (RDW) and platelet count up-trended while mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) down-trended over the 8 weeks, suggestive of an evolving iron deficiency contributing to worsening anemia. Quantitative PCR detected a 182% increase in liver EPO transcript and a 68% increase in kidney EPO transcript in the 50 mg/kg FG-4592 treated group, compared to vehicle. No changes in liver hepcidin transcription were observed in any group. ELISA analysis detected increased plasma EPO and decreased plasma hepcidin levels with increasing doses of FG-4592. None of these trends, except increases in RDW, were statistically significant. Our study is the first to characterize the effect of HIF-PHI drugs in a mouse model of SCD. Preliminary data confirm an effect of FG-4592 on HbF induction and suggest that FG-4592 may increase EPO expression and decrease hepcidin levels in SCD mice. These results highlight the potential of prolyl hydroxylase inhibition as a novel therapeutic approach for chronic anemia in SCD. Future directions include testing higher doses of FG-4592 to induce a stronger erythropoietic effect and monitoring additional markers of iron homeostasis in our mouse model of SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Palmitoylethanolamide Attenuates Pain-like Behavior in Factor VIII Deficient Mice

Coagulation factor VIII (FVIII) deficiency gives rise to hemophilia A, which is characterized by spontaneous joint bleeding. Uncontrolled bleeding drives inflammation and release of noxious and neuropathic factors, accompanied by joint degeneration, arthropathy, joint/chronic pain and impairment in physical activity. Thus, an unmet need is to develop strategies to target pain and improve physical activity in hemophilia. Chronic joint pain observed in osteoarthritis (OA) shares features of inflammation and hemarthropathy with hemophilia joint pain. Palmitoylethanolamide (PEA) is being evaluated for joint pain in OA in an interventional clinical trial (NCT05406726), which builds upon a previous double-blind, placebo-controlled evaluation suggesting its safety, tolerability, and efficacy in alleviating OA joint pain. PEA is an endogenous lipid mediator that interacts with the endogenous cannabinoid system to alleviate chronic pain, inflammation and signs of neuropathy without the intoxicating effects of cannabinoids. We reported that PEA reduced chronic pain in a mouse model of sickle cell disease (Argueta et al., Blood 2021), which presents with release of cell free heme, consequent inflammation and tissue damage similar to hemophilia. Due to its ability to reduce clinical pain outcomes and its beneficial effects on inflammation, osteoclastogenesis, and neuropathy, PEA may provide substantial benefits as a novel treatment for pain in hemophilia. To test this hypothesis, we utilized FVIII-knockout (KO) mice, which recapitulate features of human hemophilia A including spontaneous bleeding and pain (Cooke et al., J Thromb Haemost 2019), to evaluate the impact of daily PEA treatment on nociceptive and nocifensive behaviors. We treated 12 wks old male control (B6129SF2/J) and FVIII-KO (B6;129S-F8tm1Kaz/J) mice (Jackson Laboratory, Bar Harbor, ME) intraperitoneally with 20 mg/kg/d PEA (Cayman Chemical, Ann Arbor, MI) for 7 days followed by 7 day withdrawal. Mice were weighed and examined for mechanical hyperalgesia by application of Von Frey monofilaments to the plantar surface of the hindpaws (10 repetitions per paw) and weight bearing on pressure-sensitive plates (Incapacitance Tester, Ugo Basile, Gemonio, Italy) at baseline (BL), then after PEA treatment at 1, 4, and 24 hours, days 3 and 7; and after discontinuation of treatment on days 10 and 14. We also measured spontaneous nocifensive behaviors (locomotion, rearing, and grooming) over a 2-minute period in homecage (Generic USB webcam; Streampix 7, Norpix, Montreal, Canada) at BL, days 7 and 14. PEA treatment reduced mechanical hyperalgesia (~60%, P<0.001) in FVIII-KO mice after 1 hour, which continued to improve over 24 hours and was maintained for 7 days of treatment. Following withdrawal, mechanical hyperalgesia was no longer alleviated. These data suggest increased sensory pain in FVIII-KO mice, and that PEA has an analgesic effect. PEA treatment improved stance instability in weight bearing test of FVIII-KO mice (~66%, P<0.05) after 4 hours, which continued to improve over treatment period and persisted after withdrawal, up to day 10. However, improved weight bearing regressed by day 14. Improved weight bearing even after withdrawal of the PEA for 3 days suggests a disease modifying effect perhaps via reduction in joint inflammation, modulation of arthropathy and/other mechanisms. Non-evoked, spontaneous nocifensive behaviors show associations (Pearson correlation, P<0.05) with weight bearing at BL, days 7 and 14 in FVIII-KO mice. Thus, nocifensive behaviors may be a less invasive, indirect measure of weight bearing and joint pain. Body mass was not significantly affected by treatment and subsequent withdrawal. We provide the first evidence that PEA may reduce mechanical hyperalgesia and improve weight bearing in a translational model of hemophilia A. Several clinical evaluations of PEA indicate its safety, even at relatively large doses, and its efficacy in reducing inflammation and pain, which include joint pain and damage observed in OA. Our observations and the availability of PEA as a dietary supplement underscore the need for clinical and preclinical evaluation of PEA for hemophilia pain.

PRMT5 Inhibition Reduces Autoinflammation in a Murine Model of Secondary Hemophagocytic Lymphohistiocytosis

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare but frequently fatal autoinflammatory syndrome characterized by dysregulated immune activation, excessive production of proinflammatory cytokines and hyperactivation of T cells and macrophages leading to tissue damage. Primary HLH occurs due to predisposing genetic mutations in the perforin/granzyme pathway, while secondary HLH can be triggered by infections, rheumatologic disorders, or malignancies. HLH has a poor overall prognosis despite appropriate therapy, and new avenues targeting pathologic disease mechanisms are urgently needed. The protein arginine methyltransferase 5 (PRMT5) enzyme has been identified as an important mediator in inflammatory T cell subsets in autoimmune disease and graft versus host disease. We hypothesized that PRMT5 activity is relevant to inflammation loops that drive autoinflammation observed in HLH. Methods: We explored inhibition of type II protein arginine methyltransferase enzyme, PRMT5, in a murine model of secondary HLH. HLH was triggered in C57BL/6-J mice (Jackson Laboratories) by repeated injections of CpG 1826 (Integrated DNA Technologies) and an IL-10 receptor-blocking antibody (clone 1B1.3A; Bio X Cell) on days 0, 2, 4, and 7. Injected mice were left untreated or were treated with ruxolitinib 90 mg/kg (validated treatment of HLH) orally twice daily, starting on Day 4, or increasing doses of oral inhibitor of PRMT5, PRT382 (0.25, 1 or 2 mg/kg daily), starting on Day 0. Mice were weighed daily. On day 9, mice were euthanized and analyzed for spleen and liver weight, serum IFNγ levels and immune profiling of lymphoid and myeloid splenocyte subsets. Splenocytes were left unstimulated, or stimulated with lipopolysaccharide (LPS, myeloid cell stimulation) or CD3/CD28-directed antibodies (T cell stimulation) and analyzed by flow cytometry. Data analysis was performed by unsupervised ViSNE clustering in Cytobank software. Statistical analysis was performed by unpaired t test. Results: We observed that 1 mg/kg PRT382 lessened the hallmarks of secondary HLH, compared to the untreated HLH cohort, including weight loss (21% reduction, p-value 0.2746), the development of splenomegaly (71% reduction, p-value <0.001) and hepatomegaly (61% reduction, p-value <0.05), similarly to ruxolitinib (p-values <0.01, <0.001, and 0.0795, respectively). Furthermore, mice with untreated HLH showed a 17X increase in serum IFNγ levels compared to controls (p-value <0.01), while mice treated with ruxolitinib showed a 10X increase (p-value <0.05 compared to controls, 0.2577 compared to untreated HLH), and mice treated with 1 mg/kg PRT382 showed undetectable serum IFNγ (p-value 0.2104 compared to controls, <0.01 compared to untreated HLH). Flow cytometric analysis of mouse splenocytes showed that HLH stimulated the expansion of neutrophils (CD11b+/Ly6Clo/Ly6G+) and inflammatory monocytes (CD11b+/Ly6C+/Ly6G-). Ruxolitinib partially counteracted this expansion. PRT382 inhibited expansion of these myeloid cell subsets in a dose-dependent manner and also limited production of IFNγ and TNFα by myeloid cells in response to stimulation with LPS in presence of Golgi blockade with Brefeldin A. Furthermore, PRT382 limited the upregulation of CD25 on the surface of T cells in response to T cell stimulation, although no change in cytokine production by T cells was observed. Conclusions: PRMT5 inhibition counteracts pro-inflammatory features of pathologic hyper-inflammation driven by myeloid immune cell subsets in a model of secondary HLH. This may represent a novel avenue for urgently needed immune modulatory targeted treatment of HLH. Studies of PRMT5 expression and targeting in models of primary HLH are underway.