Tg Mice Research Articles

GITRL expressed by regulatory B cells is the key to expanding T-regulatory cells to the level required to ameliorate autoimmunity

Abstract CD4+Foxp3+ T regulatory cells (Treg) are well characterized for their role in controlling autoimmunity. We have previously shown that mice deficient in B cells (μMT) have significantly reduced Treg and cannot recover from the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), demonstrating a regulatory role for B cells in immune tolerance. Using B cell depletion strategies and phenotyping, we discovered B cell IgD Low (BDL) regulatory B cells that when adoptively transferred into μMT mice increased Treg numbers and drove EAE recovery. BDL activity was found to be dependent upon glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL), which engages GITR on Treg inducing their proliferation. To investigate whether BDL activity could be enhanced, B cell-specific GITRL transgenic (tg) mice were obtained. These mice had three-fold more Treg than WT mice consistent with attenuated EAE. Adoptive transfer of GITRLtg BDL into μMT mice did not increase Treg numbers nor reduce EAE severity as compared to WT BDL. Here we posit that sustaining elevated Treg numbers is required to ameliorate EAE to clinical scores below that of WT, as seen in GITRLtg mice. This holds translational implications for many autoimmune diseases for which Treg cell therapies have been proposed. New approaches must be examined to not only increase Treg in autoimmune patients, but also to retain these levels over longer periods of time.

High-Fat Diet-Induced Gut Microbiota Alterations Worsen Experimental Autoimmune Encephalomyelitis Severity

Abstract Multiple sclerosis (MS) is a complex neuroinflammatory disease caused by both genetic and environmental factors. Genetically, HLA class II alleles such as HLA-DR2 and –DR3 are strongly linked with MS. Among environmental factors, obesity and gut microbiota has emerged as an important risk factor in MS pathogenesis and severity. This study investigated the impact of High-Fat-Diet (HFD)-induced gut microbiota alterations on experimental autoimmune encephalomyelitis (EAE), utilizing HLA-DR3 (0301) and HLA-DR2(1501).DQ6(0602) transgenic (Tg) mice, crucial for investigating genetic-environment interactions. HFD induces obesity in HLA-DR3, HLA-DR2.DQ6 Tg, and C57BL/6 (B6) mice, exacerbated EAE severity and induced CNS inflammation, and demyelination. Fecal microbiota analysis revealed microbiome dysbiosis with a significant increase in hydrogen sulfide (H2S)-producing bacteria such as Desulfovibrio piger and Bilophila wadsworthia. Mono-colonization of D. piger and B. wadsworthia in gut microbiota-depleted B6 mice mirrored the heightened EAE severity observed in HFD-fed mice. Further, antibiotic depletion study in HFD-fed mice reduced EAE severity and fecal H2S levels, highlighting importance of H2S-producing gut bacteria in obesity-induced EAE modulation. This study identified a disease-promoting role of H2S-producing gut bacteria and underscored the possibility of targeting bacterial H2S as a potential therapeutic intervention to alleviate disease severity in obese MS patients.

HBV immune checkpoint receptor CD33 (Siglec-3) is a promising therapeutic target for chronic hepatitis B infection

Abstract Approximately 300 million people globally suffer from chronic hepatitis B (CHB), leading to complications like hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. Despite a significant reduction in new HBV infections through vaccination, a definitive CHB cure remains elusive. Current treatments rely on antiviral nucleos(t)ide analogues, often failing to induce crucial anti-HBsAg antibody (HBsAb) production. Our prior study unveiled myeloid CD33 (Siglec-3) as an immune checkpoint receptor for HBV, contributing to HBV-induced tolerance. Blocking CD33 with anti-hCD33 mAb enhances antigen-presenting molecule expression and activation markers in dendritic cells, restoring TLR7 agonist-induced cytokine production in CHB patient PBMCs. We recently identified a novel CD19+ B cell population expressing abundant CD33 in CHB patient PBMCs (5-20% of CD19+ B cells), contrasting with rare detection in healthy donors (0.5-3%). Anti-hCD33 mAb treatment induces HBsAb production in PBMCs and CD19+ B cells. Furthermore, establishing a CHB mouse model via pAAV-HBV-1.2 hydrodynamic injection in human CD33 transgenic mice (hCD33 Tg) demonstrated prolonged HBsAg persistence compared to wild-type mice, suggesting human CD33 impedes HBV clearance. Remarkably, anti-hCD33 mAb administration accelerates HBsAg clearance in hCD33 Tg mice, highlighting CD33 as a promising CHB therapeutic target, with anti-hCD33 mAb potential to reactivate host immunity for HBV clearance in CHB patients.

ACE2 and a Traditional Chinese Medicine Formula NRICM101 Could Alleviate the Inflammation and Pathogenic Process of Acute Lung Injury

The coronavirus disease 2019 (COVID-19) is a highly transmittable respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and acute lung injury (ALI) is the major complication of COVID-19. The challenge in studying SARS-CoV-2 pathogenicity is the limited availability of animal model. Therefore, it is necessary to establish animal models that can reproduce multiple characteristics of ALI to study therapeutic application. The present study established a mouse model that has features of ALI that are similar to COVID-19 syndrome to investigate the role of human angiotensin-converting enzyme type II (ACE2) and the administration of the Chinese herbal prescription Chingguan Yihau (NRICM101) in ALI. The mice with ACE2 genetic modifications, including the overexpression of human ACE2 (K18-hACE2 TG) and the absence of ACE2 (mACE2 KO), were intratracheally instillated with hydrochloric acid to induce ALI. The acid intratracheal instillation induced the imbalance between angiotensin-converting enzyme (ACE) and ACE2 (i.e., ACE/ACE2), severe immune cell infiltration, cytokine storms, inflammatory effect, and pulmonary disease in the mice. Compared with K18-hACE2 TG mice, mACE2 KO mice exhibited dramatically increased levels of multiple inflammatory cytokines Interleukin-6 (IL-6) and Tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid, histological evidence of lung injury, the production of the ACE expression, and the activation of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression. The results indicate that overproduction of ACE2 and induction of the ACE2-Ang-(1-7)/Mas axis could counteract the proinflammatory effect of acid-induced ALI and effectively alleviate the pathogenic process of acid-induced ALI by regulating MAPK, p-ERK1/2, and p-STAT3 pathways and dysregulation of MMP-2/MMP-9 activation. In addition, NRICM101, which is an effective TCM that has both antiviral and anti-inflammatory effects against COVID-19, shows potential for treating acid-induced ALI. NRICM101 could alleviate the disease progression of acid-induced ALI by suppressing IL-6 and TNF-α production in alveoli. In conclusion, the established mouse model provided an effective platform for researchers to investigate the role of ACE2 on pathological mechanisms and develop therapeutic strategies for ALI, including COVID-19-related pulmonary diseases. The effects of NRICM101 administration is a potential strategy for curing acid-induced ALI evaluated in the mouse model in this study. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Thbs1 regulates skeletal muscle mass in a TGFβ-Smad2/3-ATF4-dependent manner

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor β (TGFβ)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFβ-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFβ-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFβ-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.

Arbovirus infection increases the risk for the development of neurodegenerative disease pathology in the murine model

Alzheimer's disease is classified as a progressive disorder resulting from protein misfolding, also known as proteinopathies. Proteinopathies include synucleinopathies triggered by misfolded amyloid α-synuclein, tauopathies triggered by misfolded tau, and amyloidopathies triggered by misfolded amyloid of which Alzheimer's disease (β-amyloid) is most prevalent. Most neurodegenerative diseases (>90%) are not due to dominantly inherited genetic causes. Instead, it is thought that the risk for disease is a complicated interaction between inherited and environmental risk factors that, with age, drive pathology that ultimately results in neurodegeneration and disease onset. Since it is increasingly appreciated that encephalitic viral infections can have profoundly detrimental neurological consequences long after the acute infection has resolved, we tested the hypothesis that viral encephalitis exacerbates the pathological profile of protein-misfolding diseases. Using a robust, reproducible, and well-characterized mouse model for β-amyloidosis, Tg2576, we studied the contribution of alphavirus-induced encephalitis (TC-83 strain of VEEV to model alphavirus encephalitis viruses) on the progression of neurodegenerative pathology. We longitudinally evaluated neurological, neurobehavioral, and cognitive levels, followed by a post-mortem analysis of brain pathology focusing on neuroinflammation. We found more severe cognitive deficits and brain pathology in Tg2576 mice inoculated with TC-83 than in their mock controls. These data set the groundwork to investigate sporadic Alzheimer's disease and treatment interventions for this infectious disease risk factor.

DENND1A desensitizes granulosa cells to FSH by arresting intracellular FSHR transportation.

Polycystic ovary syndrome (PCOS) is a complex disorder. Genome-wide association studies (GWAS) have identified several genes associated with this condition, including DENND1A. DENND1A encodes a clathrin-binding protein that functions as a guanine nucleotide exchange factor involved in vesicular transport. However, the specific role of DENND1A in reproductive hormone abnormalities and follicle development disorders in PCOS remain poorly understood. In this study, we investigated DENND1A expression in ovarian granulosa cells (GCs) from PCOS patients and its correlation with hormones. Our results revealed an upregulation of DENND1A expression in GCs from PCOS cases, which was positively correlated with testosterone levels. To further explore the functional implications of DENND1A, we generated a transgenic mouse model overexpressing Dennd1a (TG mice). These TG mice exhibited subfertility, irregular estrous cycles, and increased testosterone production following PMSG stimulation. Additionally, the TG mice displayed diminished responsiveness to FSH, characterized by smaller ovary size, less well-developed follicles, and abnormal expressions of FSH-priming genes. Mechanistically, we found that Dennd1a overexpression disrupted the intracellular trafficking of follicle stimulating hormone receptor (FSHR), promoting its internalization and inhibiting recycling. These findings shed light on the reproductive role of DENND1A and uncover the underlying mechanisms, thereby contributing valuable insights into the pathogenesis of PCOS and providing potential avenues for drug design in PCOS treatment.

Glycan-Reactive Innate-like B Cells and Developmental Checkpoints.

Using an Ig H chain conferring specificity for N-acetyl-d-glucosamine (GlcNAc), we developed transgenic (VHHGAC39 TG) mice to study the role of self-antigens in GlcNAc-reactive B-1 B cell development. In VHHGAC39 TG mice, GlcNAc-reactive B-1 B cell development during ontogeny and in adult bone marrow was normal. However, adult TG mice exhibited a block at transitional-2 immature B cell stages, resulting in impaired allelic exclusion and accumulation of a B cell subset coexpressing endogenous Ig gene rearrangements. Similarly, VHHGAC39 B cell fitness was impeded compared with non-self-reactive VHJ558 B TG cells in competitive mixed bone marrow chimeras. Nonetheless, adult VHHGAC39 mice immunized with Streptococcus pyogenes produce anti-GlcNAc Abs. Peritoneal cavity B cells transferred from VHHGAC39 TG mice into RAG-/- mice also exhibited robust expansion and anti-GlcNAc Ab production. However, chronic treatment of young VHHGAC39 mice with GlcNAc-specific mAbs leads to lower GlcNAc-binding B cell frequencies while increasing the proportion of GlcNAc-binding B1-a cells, suggesting that Ag masking or clearance of GlcNAc Ags impedes maturation of newly formed GlcNAc-reactive B cells. Finally, BCR H chain editing promotes expression of endogenous nontransgenic BCR alleles, allowing potentially self-reactive TG B cells to escape anergy or deletion at the transitional stage of precursor B cell development. Collectively, these observations indicate that GlcNAc-reactive B cell development is sensitive to the access of autologous Ags.

Immunotherapy with an antibody against CD1d modulates neuroinflammation in an α-synuclein transgenic model of Lewy body like disease.

The neuroinflammatory process in synucleinopathies of the aging population such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) involves microglial activation as well as infiltration of the CNS by T cells and natural killer T cells (NKTs). To evaluate the potential of targeting NKT cells to modulate neuroinflammation, we treated α-syn transgenic (tg) mice (e.g.: Thy1 promoter line 61) with an antibody against CD1d, which is a glycoprotein expressed in antigen presenting cells (APCs). CD1d-presented lipid antigens activate NKT cells through the interaction with T cell receptor in NKTs, resulting in the production of cytokines. Thus, we hypothesized that blocking the APC-NKT interaction with an anti-CD1d antibody might reduce neuroinflammation and neurodegeneration in models of DLB/PD. Treatment with the anti-CD1d antibody did not have effects on CD3 (T cells), slightly decreased CD4 and increased CD8 lymphocytes in the mice. Moreover, double labeling studies showed that compared to control (IgG) treated α-syn tg mice, treatment with anti-CD1d decreased numbers of CD3/interferon γ (IFN γ)-positive cells, consistent with NKTs. Further double labeling studies showed that CD1d-positive cells co-localized with the astrocytes marker GFAP and that anti-CD1d antibody reduced this effect. While in control α-syn tg mice CD3 positive cells were near astrocytes, this was modified by the treatment with the CD1d antibody. By qPCR, levels of IFN γ, CCL4, and interleukin-6 were increased in the IgG treated α-syn tg mice. Treatment with CD1d antibody blunted this cytokine response that was associated with reduced astrocytosis and microgliosis in the CNS of the α-syn tg mice treated with CD1d antibody. Flow cytometric analysis of immune cells in α-syn tg mice revealed that CD1d-tet + T cells were also increased in the spleen of α-syn tg mice, which treatment with the CD1d antibody reduced. Reduced neuroinflammation in the anti-CD1d-treated α-syn tg mice was associated with amelioration of neurodegenerative pathology. These results suggest that reducing infiltration of NKT cells with an antibody against CD1d might be a potential therapeutical approach for DLB/PD.

Osterix-driven LINC complex disruption in vivo diminishes osteogenesis at 8 weeksbut not at 15 weeks.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a crucial connective component between the nuclear envelope and the cytoskeleton involving various cellular processes including nuclear positioning, nuclear architecture, and mechanotransduction. How LINC complexes regulate bone formation in vivo, however, is not well understood. To start bridging this gap, here we created a LINC disruption murine model using transgenic mice expressing Cre recombinase enzyme under the control of the Osterix (Osx-Cre) which is primarily active in pre-osteoblasts and floxed Tg(CAG-LacZ/EGFP-KASH2) mice. Tg(CAG-LacZ/EGFP-KASH2) mice contain a lox-STOP-lox flanked LacZ gene which is deleted upon cre recombination allowing for the overexpression of an EGFP-KASH2 fusion protein. This overexpressed protein disrupts endogenous Nesprin-Sun binding leading to disruption of LINC complexes. Thus, crossing these two lines results in an Osx- driven LINC disruption (ODLD) specific to pre-osteoblasts. In this study, we investigated how this LINC disruption affects exercise-induced bone accrual. ODLD cells had decreased osteogenic and adipogenic potential in vitro compared to non-disrupted controls and sedentary ODLD mice showed decreased bone quality at 8 weeks. Upon access to a voluntary running wheel,ODLD animals showed increased running time and distance; however, our 6-week exercise intervention did not significantly affect bone microarchitecture and bone mechanical properties.

The R436Q missense mutation in WWP1 disrupts autoinhibition of its E3 ubiquitin ligase activity, leading to self-degradation and loss of function.

Muscular dystrophy in the NH-413 chicken is caused by a missense mutation in the WWP1 gene. WWP1 is a HECT-type E3 ubiquitin ligase containing four tandem WW domains that interact with proline-rich peptide motifs of target proteins, and a short region connecting the second and third WW domains is crucial for the E3 ligase to maintain an autoinhibitory state. A mutation of the arginine in the WW2-WW3 linker to glutamine is thought to affect WWP1 function, but there is little information on this mutation to date. In this study, we generated a transgenic (Tg) mouse model expressing the WWP1 transgene with the R436Q mutation, which corresponds to the missense mutation found in the NH-413 chicken. Tg mice showed marked degradation of mutant WWP1 proteins in various tissues, particularly in striated muscle. Immunoprecipitation analysis using a WWP1-specific antibody demonstrated that the mutant WWP1 proteins lacked the C-terminal catalytic cysteine residue that is required for their binding to the E2-substrate complex during their degradation. In vitro analysis using the R436Q mutant of WWP1 lacking this catalytic cysteine residue showed no autodegradation, indicating that the loss-of-function degradation of this protein is caused by self-ubiquitination. Tg mice expressing R436Q WWP1 did not show stunted growth or premature death. Furthermore, histological analysis did not reveal any obvious changes. These observations suggested that the R436Q mutant WWP1 protein, which is released from autoinhibitory mode by its missense mutation, does not have abnormally activated enzyme function to substrates before its self-degradation and loss of enzyme function.

Rapid in vivo evaluation system for cholestasis-related genes in mice with humanized bile acid profiles.

Pediatric cholestatic liver diseases (Ped-CLD) comprise many ultrarare disorders with a genetic basis. Pharmacologic therapy for severe cases of Ped-CLD has not been established. Species differences in bile acid (BA) metabolism between humans and rodents contribute to the lack of phenocopy of patients with Ped-CLD in rodents and hinder the development of therapeutic strategies. We aimed to establish an efficient in vivo system to understand BA-related pathogenesis, such as Ped-CLD. We generated mice that express spCas9 specifically in the liver (L-Cas9Tg/Tg [liver-specific Cas9Tg/Tg] mice) and designed recombinant adeno-associated virus serotype 8 encoding small-guide RNA (AAV8 sgRNA) targeting Abcc2, Abcb11, and Cyp2c70. In humans, ABCC2 and ABCB11 deficiencies cause constitutional hyperbilirubinemia and most severe Ped-CLD, respectively. Cyp2c70 encodes an enzyme responsible for the rodent-specific BA profile. Six-week-old L-Cas9Tg/Tg mice were injected with this AAV8 sgRNA and subjected to biochemical and histological analysis. Fourteen days after the injection with AAV8 sgRNA targeting Abcc2, L-Cas9Tg/Tg mice exhibited jaundice and phenocopied patients with ABCC2 deficiency. L-Cas9Tg/Tg mice injected with AAV8 sgRNA targeting Abcb11 showed hepatomegaly and cholestasis without histological evidence of liver injury. Compared to Abcb11 alone, simultaneous injection of AAV8 sgRNA for Abcb11 and Cyp2c70 humanized the BA profile and caused higher transaminase levels and parenchymal necrosis, resembling phenotypes with ABCB11 deficiency. This study provides proof of concept for efficient in vivo assessment of cholestasis-related genes in humanized bile acid profiles. Our platform offers a more time- and cost-effective alternative to conventional genetically engineered mice, increasing our understanding of BA-related pathogenesis such as Ped-CLD and expanding the potential for translational research.

Crosstalk of lysyl oxidase-like 1 and lysyl oxidase prolongs their half-lives and regulates liver fibrosis through Notch signal.

Lysyl oxidase (LOX) family members (LOX and LOXL1 to 4) are crucial copper-dependent enzymes responsible for cross-linking collagen and elastin. Previous studies have revealed that LOX and LOXL1 are the most dramatically dysregulated LOX isoforms during liver fibrosis. However, the crosstalk between them and the underlying mechanisms involved in the profibrotic behaviors of HSCs, as well as the progression of liver fibrosis, remain unclear. pCol9GFP-HS4,5Tg mice, Loxl1fl/flGfapCre mice, human HSC line, and primary HSCs were enrolled to study the dysregulation pattern, profibrotic roles, and the potential mechanisms of LOX and LOXL1 interaction involved in the myofibroblast-like transition of HSCs and liver fibrogenesis. LOX and LOXL1 were synergistically upregulated during liver fibrogenesis, irrespective of etiology, together orchestrating the profibrotic behaviors of HSCs. LOX and LOXL1 coregulated in HSCs, whereas LOXL1 dominated in the coregulation loop. Interestingly, the interaction between LOXL1 and LOX prolonged their half-lives, specifically enhancing the Notch signal-mediated myofibroblast-like transition of HSCs. Selective disruption of Loxl1 in Gfap+ HSCs deactivated the Notch signal, inhibited HSC activation, and relieved carbon tetrachloride-induced liver fibrosis. Our current study confirmed the synergistic roles and the underlying mechanisms of LOXL1 and LOX crosstalk in the profibrotic behaviors of HSCs and liver fibrosis progression, providing experimental evidence for further clear mechanism-based anti-LOXL1 strategy development in the therapy of liver fibrosis.

Genotype- and sex-specific changes in vital parameters during isoflurane anesthesia in a mouse model of Alzheimer's disease.

The prevalence of neurodegenerative diseases is increasing as is life expectancy with Alzheimer's disease accounting for two-thirds of dementia cases globally. Whether general anesthesia and surgery worsen cognitive decline is still a matter of debate and most likely depending on the interplay of various influencing factors. In order to account for this complexity, Alzheimer's disease animal models have been developed. The Tg2576 model of Alzheimer's disease is a well-established mouse model exhibiting amyloidopathy and age-dependent sex-specific differences in Alzheimer's disease symptomology. Yet, data on anesthesia in this mouse model is scarce and a systematic comparison of vital parameters during anesthesia with wild-type animals is missing. In order to investigate the safety of general anesthesia and changes in vital parameters during general anesthesia in Tg2576 mice, we did a secondary analysis of vital parameters collected during general anesthesia in aged Tg2576 mice. After governmental approval (General Administration of the Free State of Bavaria, file number: 55.2-1-54-2532-149-11) 60 mice at 10-12 months of age were exposed to isoflurane (1.6 Vol%) for 120 min, data of 58 mice was analyzed. During general anesthesia, heart rate, respiratory rate, temperature, isoflurane concentration and fraction of inspired oxygen were monitored and collected. Data were analyzed using univariate and multivariate linear mixed regression models. During general anesthesia, heart rate decreased in a sex-specific manner. Respiratory rate decreased and body temperature increased dependent on genotype. However, the changes were limited and all vital parameters stayed within physiological limits. Isoflurane anesthesia in the Tg2576 mouse model is safe and does not seem to influence experimental results by interacting with vital parameters. The present study provides information on appropriate anesthesia in order to advance research on anesthesia and AD and could contribute to improving laboratory animal welfare.

Non-neuronal cholinergic system in the heart influences its homeostasis and an extra-cardiac site, the blood-brain barrier.

The non-neuronal cholinergic system of the cardiovascular system has recently gained attention because of its origin. The final product of this system is acetylcholine (ACh) not derived from the parasympathetic nervous system but from cardiomyocytes, endothelial cells, and immune cells. Accordingly, it is defined as an ACh synthesis system by non-neuronal cells. This system plays a dispensable role in the heart and cardiomyocytes, which is confirmed by pharmacological and genetic studies using murine models, such as models with the deletion of vesicular ACh transporter gene and modulation of the choline acetyltransferase (ChAT) gene. In these models, this system sustained the physiological function of the heart, prevented the development of cardiac hypertrophy, and negatively regulated the cardiac metabolism and reactive oxygen species production, resulting in sustained cardiac homeostasis. Further, it regulated extra-cardiac organs, as revealed by heart-specific ChAT transgenic (hChAT tg) mice. They showed enhanced functions of the blood-brain barrier (BBB), indicating that the augmented system influences the BBB through the vagus nerve. Therefore, the non-neuronal cardiac cholinergic system indirectly influences brain function. This mini-review summarizes the critical cardiac phenotypes of hChAT tg mice and focuses on the effect of the system on BBB functions. We discuss the possibility that a cholinergic signal or vagus nerve influences the expression of BBB component proteins to consolidate the barrier, leading to the downregulation of inflammatory responses in the brain, and the modulation of cardiac dysfunction-related effects on the brain. This also discusses the possible interventions using the non-neuronal cardiac cholinergic system.

Abstract 114: Identification of the actionable target, LILRB4, through genetic linkage analysis of Diversity Outbred (DO) F1 mice expressing HER2(neu)

Abstract Our goal is to identify, in situ, the regulatory genes that dictate the onset age and growth rate of spontaneously arising tumors to implement new intervention strategies. For genetic linkage analysis, we crossed HER2(neu) Tg mice with Diversity Outbred (DO) mice, an outbred population comprised of 8 founding strains (A/J, C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, WSB/EiJ) maintained by non-sibling crossing to ensure each mouse is genetically unique. Using R/QTL package, we linked Quantitative Trait Loci (QTL) in Chr 1 and X with tumor onset age, and a Chr 10 QTL with tumor growth rate. The Chr1 (=human Chr2) QTL was linked to human breast cancer diagnosis in 11 Genome-Wide Association Studies (GWAS; the human GWAS catalog, https://www.ebi.ac.uk/gwas/). Because human GWAS data is deficient in Chr X mapping and also does not assess tumor growth rate as a trait, the QTL loci identified in mouse Chr X and 10 were discoveries beyond the capacity of human GWAS. In total, we identified 26 candidate genes across the 3 QTL. For clinical validation, the genes were analyzed with CodeAI (Caris LS) which associated cancer patient clinical data with candidate gene expression. We found 21/26 genes significantly associated with the survival of patients with primary (n=3,533) and/or metastatic (n=4,870) breast cancers and 17/26 were associated with lung cancer (n=11,334) survival, with 13 overlapping genes between lung and breast cancer, indicating broad applicability of these candidate genes. We interrogated the immune status in BALB NeuT and (BALBxPWK)F1 NeuT mice because the F1 mice developed tumors around 9 wk, much earlier than the 14 wk observed in BALB NeuT mice. Surprisingly, (BALBxPWK)F1 mice responded to cancer vaccines more vigorously. Single cell RNA (scRNA) libraries from (BALBxPWK)F1 NeuT tumors showed an expanded lymphocyte cluster (12%), compared with BALB NeuT (2%) and an enrichment of T cell activation genes. These findings may reflect a deficiency in tumor cell recognition by the activated anti-tumor effectors and a window of opportunity for correction. Of the 21 candidate genes, LILRB4, a myeloid cell checkpoint molecule, was of particular interest. Expression of LILRB4 is predictive of patient survival in both breast and lung cancer. Using scRNA sequencing, we found this transmembrane receptor expressed primarily by tumor infiltrating myeloid cells. In a reported scRNA sequencing dataset (GSE161529), LILRB4 was expressed by infiltrating macrophages in human breast cancers, and is also correlated with reduced survival. In a PAN Cancer ATACseq dataset (Corces et al., 2018), promoter accessibility of LILRB4 was associated with reduced survival. Taken together, there are multiple immune regulatory mechanisms of tumor progression and LILRB4 is an important new actionable target. CA76340 (WZW), KCI TBM program (JBJ) and CCSG P30 CA022453 (GB). Citation Format: Jennifer B. Jacob, Wei-Zen Wei, Benjamin L. Kidder, Tolulope Adeyelu, Andrew Elliott, Gerold Bepler, Joyce D. Reyes. Identification of the actionable target, LILRB4, through genetic linkage analysis of Diversity Outbred (DO) F1 mice expressing HER2(neu) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 114.

Development and preclinical evaluation of equine-derived hyperimmune serum against SARS-CoV-2 infection in K-18 hACE2 transgenic (Tg) mice.

This study aimed to develop an equine-derived hyperimmune serum against SARS-CoV-2 and evaluate its efficacy as a potential immunotherapy tool for the treatment of known and potential variants of COVID-19 in preclinical trials. The novelty of this study is the whole virus and ALUM gel adjuvant formula. The horses were immunized using a whole inactivated SARS-CoV-2 antigen, and the final purified hyperimmune serum showed high plaque reduction neutralization (PRNT 50) neutralizing titers. The efficacy of the hyperimmune serum was evaluated histopathologically and biochemically in the lungs, hearts, and serum of K18 hACE2 transgenic mice (n=45), which is an accepted model organism for SARS-CoV-2 studies and was challenged with live SARS-CoV-2. Serum treatment improved the general condition, resulting in lower levels of proinflammatory cytokines in the blood plasma, as well as reduced viral RNA titers in the lungs and hearts. Additionally, it reduced oxidative stress significantly and lessened the severity of interstitial pneumonia in the lungs when compared to infected positive controls. The study concluded that equine-derived anti-SARS-CoV-2 antibodies could be used for COVID-19 prevention and treatment, especially in the early stages of the disease and in combination with antiviral drugs and vaccines. This treatment will benefit special patient populations such as immunocompromised individuals, as specific antibodies against SARS-CoV-2 can neutralize the virus before it enters host cells. The rapid and cost-effective production of the serum allows for its availability during the acute phase of the disease, making it a critical intervention in preventing the spread of the disease and saving lives in new variants where a vaccine is not yet developed.

Artificial intelligence-driven drug repositioning uncovers efavirenz as a modulator of α-synuclein propagation: Implications in Parkinson’s disease

Parkinson's disease (PD) is a complex neurodegenerative disorder with an unclear etiology. Despite significant research efforts, developing disease-modifying treatments for PD remains a major unmet medical need. Notably, drug repositioning is becoming an increasingly attractive direction in drug discovery, and computational approaches offer a relatively quick and resource-saving method for identifying testable hypotheses that promote drug repositioning. We used an artificial intelligence (AI)-based drug repositioning strategy to screen an extensive compound library and identify potential therapeutic agents for PD. Our AI-driven analysis revealed that efavirenz and nevirapine, approved for treating human immunodeficiency virus infection, had distinct profiles, suggesting their potential effects on PD pathophysiology. Among these, efavirenz attenuated α-synuclein (α-syn) propagation and associated neuroinflammation in the brain of preformed α-syn fibrils-injected A53T α-syn Tg mice and α-syn propagation and associated behavioral changes in the C. elegans BiFC model. Through in-depth molecular investigations, we found that efavirenz can modulate cholesterol metabolism and mitigate α-syn propagation, a key pathological feature implicated in PD progression by regulating CYP46A1. This study opens new avenues for further investigation into the mechanisms underlying PD pathology and the exploration of additional drug candidates using advanced computational methodologies.

Relationship Between Reactive Astrocytes, by [18F]SMBT-1 Imaging, with Amyloid-Beta, Tau, Glucose Metabolism, and TSPO in Mouse Models of Alzheimer’s Disease

Reactive astrocytes play an important role in the development of Alzheimer’s disease (AD). Here, we aimed to investigate the temporospatial relationships among monoamine oxidase-B, tau and amyloid-β (Aβ), translocator protein, and glucose metabolism by using multitracer imaging in AD transgenic mouse models. Positron emission tomography (PET) imaging with [18F]SMBT-1 (monoamine oxidase-B), [18F]florbetapir (Aβ), [18F]PM-PBB3 (tau), [18F]fluorodeoxyglucose (FDG), and [18F]DPA-714 (translocator protein) was carried out in 5- and 10-month-old APP/PS1, 11-month-old 3×Tg mice, and aged-matched wild-type mice. The brain regional referenced standard uptake value (SUVR) was computed with the cerebellum as the reference region. Immunofluorescence staining was performed on mouse brain tissue slices. [18F]SMBT-1 and [18F]florbetapir SUVRs were greater in the cortex and hippocampus of 10-month-old APP/PS1 mice than in those of 5-month-old APP/PS1 mice and wild-type mice. No significant difference in the regional [18F]FDG or [18F]DPA-714 SUVRs was observed in the brains of 5- or 10-month-old APP/PS1 mice or wild-type mice. No significant difference in the SUVRs of any tracer was observed between 11-month-old 3×Tg mice and age-matched wild-type mice. A positive correlation between the SUVRs of [18F]florbetapir and [18F]DPA-714 in the cortex and hippocampus was observed among the transgenic mice. Immunostaining validated the distribution of MAO-B and limited Aβ and tau pathology in 11-month-old 3×Tg mice; and Aβ deposits in brain tissue from 10-month-old APP/PS1 mice. In summary, these findings provide in vivo evidence that an increase in astrocyte [18F]SMBT-1 accompanies Aβ accumulation in APP/PS1 models of AD amyloidosis.

Altered Fhod3 expression involved in progressive high-frequency hearing loss via dysregulation of actin polymerization stoichiometry in the cuticular plate.

Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer hair cells, and a decreased phalloidin intensities of CP. Ultrastructural analysis revealed loss of the shortest row of stereocilia in the basal turn of the cochlea, and alterations in the cuticular plate surrounding stereocilia rootlets. Importantly, the hearing and HC phenotype in TG mice phenocopied that of the KO mice. These findings suggest that balanced expression of Fhod3 is critical for proper CP and stereocilia structure and function. Further investigation of Fhod3 related hearing impairment mechanisms may lend new insight towards the myriad mechanisms underlying ARHL, which in turn could facilitate the development of therapeutic strategies for ARHL.