Littermate Mice Research Articles

The TMEM43 S358L mutation affects cardiac, small intestine, and metabolic homeostasis in a knock-in mouse model.

The transmembrane protein 43 (TMEM43/LUMA) p.S358L mutation causes arrhythmogenic cardiomyopathy named as ARVC5, a fully penetrant disease with high risk of ventricular arrhythmias, sudden death, and heart failure. Male gender and vigorous exercise independently predicted deleterious outcome. Our systems genetics analysis revealed the importance of Tmem43 for cardiac and metabolic pathways associated with elevated lipid absorption from small intestine. This study sought to delineate gender-specific cardiac, intestinal, and metabolic phenotypes in vivo and investigate underlying pathophysiological mechanisms of S358L mutation. Serial echocardiography, surface electrocardiography (ECG), treadmill running, and body EchoMRI have been used in knock-in heterozygous (Tmem43WT/S358L), homozygous (Tmem43S358L), and wildtype (Tmem43WT) littermate mice. Electron microscopy, histology, immunohistochemistry, transcriptome, and protein analysis have been performed in cardiac and intestinal tissues. Systolic dysfunction was apparent in 3-mo-old Tmem43S358L and 6-mo-old Tmem43WT/S358L mutants. Both mutant lines displayed intolerance to acute stress at 6 mo of age, arrhythmias, fibro-fatty infiltration, and subcellular abnormalities in the myocardium. Microarray analysis found significantly differentially expressed genes between left ventricular (LV) and right ventricular (RV) myocardium. Mutants displayed diminished PPARG activities and significantly reduced TMEM43 and β-catenin expression in the heart, whereas junctional plakoglobin (JUP) translocated into nuclei of mutant cardiomyocytes. Conversely, elongated villi, fatty infiltration, and overexpression of gut epithelial proliferation markers, β-catenin and Ki-67, were evident in small intestine of mutants. We defined Tmem43 S358L-induced pathological effects on cardiac and intestinal homeostasis via distinctly disturbed WNT-β-catenin and PPARG signaling thereby contributing to ARVC5 pathophysiology. Results suggest that cardiometabolic assessment in mutation carriers may be important for predictive and personalized care.NEW & NOTEWORTHY This manuscript describes the findings of our investigation of cardiac, small intestine, and metabolic features of Tmem43-S358L mouse model. By investigating interorgan pathologies, we uncovered multiple mechanisms of the S358L-induced disease, and these unique mechanisms likely appear to contribute to the disease pathogenesis. We hope our findings are important and novel and open new avenues in the hunting for additional diagnostic and therapeutic targets in subjects carrying TMEM43 mutation.

166 The Protective Role of GPR39 in Subarachnoid Hemorrhage

INTRODUCTION: Aneurysmal subarachnoid hemorrhage (SAH) is a devastating stroke with high morbidity and mortality. It causes an early brain injury (EBI), the severity of which can predict delayed cerebral ischemia (DCI). Neuroinflammation is a key component of EBI. The G protein-coupled receptor 39 (GPR39) is expressed in brain, and it has been shown to modulate neuroinflammation. METHODS: Endovascular perforation technique induced SAH in GPR39 knockout (KO) and wild-type (WT) littermate mice. Laser-Doppler was used to monitor cortical perfusion before, during, and right after SAH. Body mass and neurological deficit (neuroscore) were assessed daily after SAH. T2-weighted MRI was used to measure ventricular volumes, and T2*-weighted gradient recalled echo (GRE) MRI was used to assess cortical vein engorgement at 24 hours after SAH. Behavioral deficit was assessed using the accelerating rotarod at baseline and on days 2, 3 and 4 after SAH and balance beam on days 2, 3 and 4 after SAH. RESULTS: SAH caused an acute, communicating hydrocephalus with engorged cortical veins. WT and KO mice exhibited significant weight loss and deficits in neuroscore after SAH compared to sham mice. GPR39 KO mice had a significantly worse neuroscore than WT mice 3 days after SAH (mean 20.7 vs. 22.3; n = 12 vs. 15, respectively; p = 0.01). Additionally, GPR39 KO mice exhibited worse scores on rotarod 2 days after SAH (mean latency to fall 138.9 vs. 108.8; n=12 vs 15, respectively, p = 0.07) and balance beam 1 day after SAH (mean time to traverse 24.8 vs. 31.8; n = 8 vs. 11, respectively, p = 0.1). CONCLUSIONS: Our findings indicate that mice with GPR39 deletion exhibit worse outcomes in neuroscore and sensorimotor domains. These results suggest that GPR39 plays a protective role and may serve as a therapeutic target in SAH.

381 The role of leucine-rich PPR motif-containing protein (LRPPRC) in myelin lipid metabolism

OBJECTIVES/GOALS: Leigh Syndrome, French Canadian-Type (LSFC) is a neurometabolic disorder caused by mutation of mitochondria-related gene, LRPPRC. White matter lesions and demyelination in central nervous system are common in LSFC. LRPPRC is enriched in myelinating glial cells, yet its role is not known. Our goal is to elucidate its mechanistic role in myelination. METHODS/STUDY POPULATION: We crossed C57BL/6N mice bearing a LRPPRC-loxP allele with mice bearing a Plp-CreERT2 allele. Mice with the Plp-CreERT2 allele expresses a tamoxifen-inducible Cre under the control of the Plp promoter, which drives expression in oligodendrocytes. Using these strains, we can target the deletion of LRPPRC, via tamoxifen injection, in both newly formed myelin and mature myelin. Plp-CreERT2; LRPPRCL/L (LRPPRC-KO) or control littermate mice will be injected for LRPPRC deletion at developmental and maturation stages of myelin. Immunofluorescence and electron microscopy of isolated brain tissues will be used for myelin integrity analysis. Cognitive functions of the mice will be measured via behavioral tests. Lastly, we will submit tissues for lipidomic analyses to observe any lipid metabolite variation. RESULTS/ANTICIPATED RESULTS: Behavioral and motor defects would be expected in LRPPRC-KO mice performing in cognitive function tasks across myelin maturation stages. Electron microscopy-based structure analysis of optic nerve, corpus callosum, and spinal cord should reveal thin or loss of myelin on the axons of LRPPRC-KO compared to control. Immunofluorescence staining of major myelin structural proteins, including myelin proteolipid protein (PLP), myelin basic protein (MBP), and myelin-associated glycoprotein (MAG) would be expected have lower levels in LRPPRC deficient tissues. Since myelin is a lipid-rich species, we would also expect lipid concentrations to be affected. LRPPRC-KO lipidomic analyses of myelin-related lipids should depict lower levels in comparison to control, which would imply dysfunctional lipid metabolism. DISCUSSION/SIGNIFICANCE: There are limited studies in ameliorating neural deficits caused by LS and LSFC. Successful completion of this project would help elucidate the functions of LRPPRC in myelination and lipid metabolism and potentially provide insights for developing novel therapeutic strategies for alleviating the demyelination and neural deficits in LSFC.

Orexin induces the production of an endocannabinoid-derived lysophosphatidic acid eliciting hypothalamic synaptic loss in obesity

ObjectiveOrexin-A (OX-A) is a neuropeptide produced selectively by neurons of the lateral hypothalamus. It exerts powerful control over brain function and physiology by regulating energy homeostasis and complex behaviors linked to arousal. Under conditions of chronic or acute brain leptin signaling deficiency, such as in obesity or short-term food deprivation, respectively, OX-A neurons become hyperactive and promote hyperarousal and food seeking. However, this leptin-dependent mechanism is still mostly unexplored. The endocannabinoid 2-arachidonoyl-glycerol (2-AG) is known to be implicated in food consumption by promoting hyperphagia and obesity, and we and others demonstrated that OX-A is a strong inducer of 2-AG biosynthesis. Here, we investigated the hypothesis that, under acute (6 h fasting in wt mice) or chronic (in ob/ob mice) hypothalamic leptin signaling reduction, OX-A-induced enhancement of 2-AG levels leads to the production of the 2-AG-derived 2-arachidonoyl-sn-glycerol-3-phosphate (2-AGP), a bioactive lipid belonging to the class of lysophosphatidic acids (LPAs), which then regulates hypothalamic synaptic plasticity by disassembling α-MSH anorexigenic inputs via GSK-3β-mediated Tau phosphorylation, ultimately affecting food intake. MethodsWe combined cell-type-specific morphological (CLEM and confocal microscopy), biochemical, pharmacological, and electrophysiological techniques to dissect the leptin- and OX-A/2-AGP-mediated molecular pathways regulating GSK-3β-controlled pT231-Tau production at POMC neurons of obese ob/ob and wild-type (wt) lean littermate mice and in an in vitro model of POMC neurons such as mHypoN41 neurons (N41). Results2-AGP is overproduced in the hypothalamus of obese leptin-deficient, or lean 6 h food-deprived mice, and promotes food intake by reducing α-MSH-expressing synaptic inputs to OX-A neurons via lysophosphatidic acid type-1 receptor (LPA1-R) activation, and pT231-Tau accumulation in α-MSH projections. This effect is due to the activation of the Pyk2-mediated pTyr216-GSK3β pathway and contributes to further elevating OX-A release in obesity. Accordingly, we found a strong correlation between OX-A and 2-AGP levels in the serum of obese mice and of human subjects. ConclusionsHypothalamic feeding pathways are endowed with 2-AGP-mediated synaptic plasticity according to their inherent functional activities and the necessity to adapt to changes in the nutritional status. These findings reveal a new molecular pathway involved in energy homeostasis regulation, which could be targeted to treat obesity and related disturbances.

Detection of early proteomic alterations in 5xFAD Alzheimer's disease neonatal mouse model via MALDI-MSI.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder, characterized by memory deficit and dementia. AD is considered a multifactorial disorder where multiple processes like amyloid-beta and tau accumulation, axonal degeneration, synaptic plasticity, and autophagic processes plays an important role. In this study, the spatial proteomic differences in the neonatal 5xFAD brain tissue were investigated using MALDI-MSI coupled to LC-MS/MS, and the statistically significantly altered proteins were associated with AD. Thirty-five differentially expressed proteins (DEPs) between the brain tissues of neonatal 5xFAD and their littermate mice were detected via MALDI-MSI technique. Among the 35 proteins identified, 26 of them were directly associated with AD. Our results indicated a remarkable resemblance in the protein expression profiles of neonatal 5xFAD brain when compared to AD patient specimens or AD mouse models. These findings showed that the molecular alterations in the AD brain existed even at birth and that some proteins are neurodegenerative presages in neonatal AD brain. HIGHLIGHTS: Spatial proteomic alterations in the 5xFAD mouse brain compared to the littermate. 26 out of 35 differentially expressed proteins associated with Alzheimer's disease (AD). Molecular alterations and neurodegenerative presages in neonatal AD brain. Alterations in the synaptic function an early and common neurobiological thread.

A60 DOWN WITH THE SHP-1: CHRONIC HELMINTH INFECTION DISRUPTS BILE ACID HOMEOSTASIS AND SIGNALLING RECEPTOR ACTIVATION IN THE MURINE SMALL INTESTINE TO POTENTIALLY IMPACT GUT IMMUNITY

Abstract Background Helminths (parasitic worms) use immunomodulatory mechanisms to maintain their presence in the host intestines. Understanding how helminths reduce inflammation in the guts may lead to new therapeutic options for inflammatory bowel diseases. Our lab uses a helminth which infects the proximal small intestine of mice to identify new, potentially immunomodulatory molecules. In this project, we examined the impact of helminth infection on bile acids (BA)s; a class of metabolites that can function as immunomodulatory signalling molecules. To our knowledge, how helminth infection alters BA homeostasis, and the potential consequences for proximal small intestine immunity have not yet been reported. Purpose Our aims were to determine whether helminth infection alters (1) the intestinal BA pool, (2) expression of BA transporters in the small intestine, and (3) expression and/or activation of the Farnesoid X receptor (FXR), a BA signalling receptor. Method We used a murine model system to study the effects of the mouse-specific helminth Heligmosomoides polygyrus on BA homeostasis, BA transporter gene expression, and BA signalling receptors in the small intestine. Male and female littermate mice were used. Targeted metabolomics was used to assess the composition of the BA pool in the small intestine. Reverse transcription qPCR was used to quantify gene expression of BA transporters/signalling molecules in tissues. All data were segregated by sex of mice for data analysis. Result(s) We found that helminth infection disrupts the BA pool of mice, with consistent reductions in T-CDCA and T-αMCA during helminth infection. In the proximal small intestine, expression of the genes that encode the apical sodium dependant BA transporter (ASBT) and organic solute transporter alpha (OSTα) were unaffected during helminth infection, however expression of the organic solute transporter β (OSTβ) gene, which encodes half of the basal BA transporter, was decreased. Expression of Shp-1, an indicator of activation of BA receptor FXR, and expression of the Fxr gene were significantly decreased in both the proximal and distal small intestine. Lastly, expression of the gene encoding Gpbar1 trended upwards in the proximal and distal small intestine of female mice. Conclusion(s) Infection of mice with a proximal small intestine-dwelling helminth impacts BA homeostasis in the small intestine. Helminth infection decreases expression of a BA transporter in the proximal small intestine, although whether this contributes to or is caused by perturbations in the BA pool during infection remains to be seen. The downregulation of BA signalling we see during helminth infection in the proximal small intestine points to a potential functional impact on the host immune response which we now intend to explore. Understanding the immunomodulatory mechanisms used by helminths to dampen the host immune response could lead to further advancements in treating intestinal inflammatory disorders such as Crohn’s and Colitis. Disclosure of Interest None Declared

Abstract P5-15-01: Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis

Abstract Breast cancer is the second leading cause of death for women in the United States. However, only 10% of breast cancers have been linked to inherited genomic mutations. Thus, identifying biomarkers to predict cancer progression and metastasis remains a clear need in the research and medical world. Here, we report a novel role for Interferon Regulatory Factor 5 (IRF5) in mammary gland development, tumorigenesis, and metastasis. Historically, IRF5 has been studied as a transcription factor in the context of genetic risk for autoimmune diseases. However, mining of The Cancer Genome Atlas revealed that loss of IRF5 expression in human breast cancer is significantly linked with progression to high grade carcinoma, increased metastasis, and decreased overall and recurrence-free survival. Supporting these analyses, we demonstrated that female Irf5-/- BALB/c mice have higher incidence of spontaneous atypical ductal hyperplasia (ADH), increased progression to DCIS, and ultimately, increased incidence of IDC. Using qPCR, FISH and IHC, we confirmed that IRF5 is expressed in both luminal and basal myoepithelial cells, but expression is higher in basal cells. Histologic analysis of whole mount preparations of Irf5-/- mammary glands revealed aberrant ductal morphogenesis, characterized by expansion of luminal and basal myoepithelial cells with a loss of organized glandular structure. RNAseq of primary mammary epithelial cells from wild type and Irf5-/- littermate mice showed Irf5-/- mammary epithelial cells to be enriched in ribosome biogenesis pathways, the physiologic consequences of which were demonstrated through increased rates of protein synthesis. Transferring our studies in vivo, we demonstrated that loss of tumor IRF5 expression resulted in decreased tumor-infiltrating lymphocytes and increased pulmonary metastasis in the murine orthotopic 4T1 implantation model. Mechanistically, we found that—as in our studies utilizing primary mammary epithelial cells—IRF5 expression in tumors regulated protein translation and ribosome biogenesis. In light of these findings, we propose IRF5 as a novel prognostic biomarker, loss of which alters mammary gland development, drives tumor initiation and metastasis, and dysregulates mammary epithelial cell protein synthesis. Citation Format: Betsy Barnes, Zarina Brune, Matthew Rice, Carter Somerville. Rediscovering IRF5: A prognostic indicator with novel roles in mammary gland development, ribosome biogenesis, tumor initiation, and metastasis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-15-01.

High-Energy, Whole-Body Proton Irradiation Differentially Alters Long-Term Brain Pathology and Behavior Dependent on Sex and Alzheimer’s Disease Mutations

Whole-body exposure to high-energy particle radiation remains an unmitigated hazard to human health in space. Ongoing experiments at the NASA Space Radiation Laboratory and elsewhere repeatedly show persistent changes in brain function long after exposure to simulations of this unique radiation environment, although, as is also the case with proton radiotherapy sequelae, how this occurs and especially how it interacts with common comorbidities is not well-understood. Here, we report modest differential changes in behavior and brain pathology between male and female Alzheimer's-like and wildtype littermate mice 7-8 months after exposure to 0, 0.5, or 2 Gy of 1 GeV proton radiation. The mice were examined with a battery of behavior tests and assayed for amyloid beta pathology, synaptic markers, microbleeds, microglial reactivity, and plasma cytokines. In general, the Alzheimer's model mice were more prone than their wildtype littermates to radiation-induced behavior changes, and hippocampal staining for amyloid beta pathology and microglial activation in these mice revealed a dose-dependent reduction in males but not in females. In summary, radiation-induced, long-term changes in behavior and pathology, although modest, appear specific to both sex and the underlying disease state.

Increased sucrose consumption in mice gene-targeted for Vmat2 selectively in NeuroD6-positive neurons of the ventral tegmental area.

Ventral tegmental area (VTA) dopamine (DA) neurons are implicated in reward processing, motivation, reward prediction error, and in substance use disorder. Recent studies have identified distinct neuronal subpopulations within the VTA that can be clustered based on their molecular identity, neurotransmitter profile, physiology, projections and behavioral role. One such subpopulation is characterized by expression of the NeuroD6 gene, and projects primarily to the nucleus accumbens medial shell. We recently showed that optogenetic stimulation of these neurons induces real-time place preference while their targeted deletion of the Vmat2 gene caused altered response to rewarding substances, including ethanol and psychostimulants. Based on these recent findings, we wanted to further investigate the involvement of the NeuroD6-positive VTA subpopulation in reward processing. Using the same NeuroD6Cre+/wt ;Vmat2flox/flox mice as in our prior study, we now addressed the ability of the mice to process sucrose reward. In order to assess appetitive behavior and motivation to obtain sucrose reward, we tested conditional knockout (cKO) and control littermate mice in an operant sucrose self-administration paradigm. We observed that cKO mice demonstrate higher response rates to the operant task and consume more sucrose rewards than control mice. However, their motivation to obtain sucrose is identical to that of control mice. Our results highlight previous observations that appetitive behavior and motivation to obtain rewards can be served by distinct neuronal circuits, and demonstrate that the NeuroD6 VTA subpopulation is involved in mediating the former, but not the latter. Together with previous studies on the NeuroD6 subpopulation, our findings pinpoint the importance of unraveling the molecular and functional role of VTA subpopulations in order to better understand normal behavior and psychiatric disease.

Decreased levels of s-nitrosylated Cx43 prevent arrhythmogenicity and myocardial infarction upon cardiac stress in duchenne muscular dystrophy.

Connexin43 (Cx43) plays a critical role in action potential propagation and cardiac contractility. In healthy cardiomyocytes, Cx43 is located at the intercalated disk, however, in cardiac diseases such as hypertrophic and dilated cardiomyopathy, it is remodelled to the lateral side of ventricular cardiomyocytes. In Duchenne muscular dystrophy (DMD) mice, lateralized cardiac Cx43 is found forming hemichannels at the plasma membrane that activate upon cardiac stress evoking triggered activity and arrhythmogenic behaviours. S-nitrosylated levels of Cx43 strongly correlate with changes in membrane excitability in cardiomyocytes from DMD mice. To unequivocally demonstrate a direct role of S-nitrosylated Cx43 in DMD cardiomyopathy, we developed a specific knockin mouse line, in which the Cx43 site for S-nitrosylation, cysteine 271, was specifically substituted for a serine (Cx43C271S). We then crossed the C271S mouse line with DMD mice to obtain DMD:Cx43C271S+/- mice with reduced levels of S-nitrosylated Cx43. Littermate mice (4-6-month-old) were used to assess isoproterenol-induced cardiac stress and, consequently, heart dysfunction in WT, DMD and DMD:Cx43C271S+/- mice. DMD mice displayed increased number of arrhythmogenic events, but without changes in RR and QRS complexes within 1-h post-isoproterenol treatment. However, elevated number of deadly arrhythmias, bradycardia, and increased QRS complex duration were detected in DMD mice for 24-h following isoproterenol stimulation. Strikingly, the number of arrhythmogenic events were significantly reduced in DMD:Cx43C271S+/- mice. β-adrenergic stimulation with isoproterenol induced cardiac hypertrophy, myocardial infarction, and 40% mortality in DMD mice, which was prevented in DMD:Cx43C271S+/- mice. Therefore, these findings strongly suggest that S-nitrosylation of Cx43 proteins at site cysteine 271 represents a fundamental nitric oxide-mediated mechanism involved in the induction of arrythmias and myocardial infarction in DMD mice after β-adrenergic stress.

BUTYRATE AMELIORATES PANETH CELL DEFECTS, TOTAL INFLAMMATION, AND EPITHELIAL CELL APOPTOSIS DURING MITOCHONDRIAL DYSFUNCTION DRIVEN BY PROHIBITIN 1-DEFICIENCY

Abstract INTRODUCTION Prohibitin 1 (PHB1) is a mitochondrial chaperone protein important for maximal activity of the electron transport chain. PHB1 deficiency in intestinal epithelial cells (IECs) or specifically in Paneth cells (PCs) induces epithelial mitochondrial dysfunction, PC defects, gut microbiota dysbiosis, and spontaneous ileitis in mice by 20 weeks-of-age. Dysbiotic gut microbiota has been implicated in inflammatory bowel diseases and is associated with the decreased abundance of short chain fatty acids (SCFAs; acetate, propionate, and butyrate)-producing bacteria. SCFAs play a significant role in reducing intestinal inflammation and epithelial barrier dysfunction. We tested the hypothesis that luminal SCFA levels were decreased in mice with PHB1 deficiency and that supplementation of SCFAs will ameliorate intestinal inflammation during mitochondrial dysfunction driven by loss of PHB1. METHODS Ileal luminal contents were collected from Phb1fl/fl (control) and Phb1iΔIEC (Phb1-deficient in IECs) mice at 20 weeks of age and SCFAs were measured by gas chromatography-mass spectrometry. Phb1iΔIEC and Phb1fl/fl littermate mice were treated with 20 mM butyrate dissolved in their drinking water from 16 to 20 weeks-of-age and PC defects were measured by antimicrobial peptide expression (Lysozyme, Cryptdin 3, Cryptdin 5, and Ang4) and Lysozyme staining allocation into secretory granules. Ileitis was measured by histological scoring and pro-inflammatory cytokine expression. To test epithelial intrinsic responses, ileal crypt enteroids derived from Phb1iΔIEC and Phb1fl/fl littermate mice were treated with 200 µg/ml healthy ileal stool lysates (from Phb1fl/fl mice) or dysbiotic stool lysates (from Phb1iΔIEC mice) for 16 hours with or without 0.05 mM butyrate, 0.5 mM propionate, 0.5 mM acetate, alone or in combination. Enteroid viability and apoptosis were measured by morphological change and cleaved Caspase 3 expression. RESULTS Butyrate was significantly decreased in ileal stool of Phb1iΔIEC mice. Butyrate supplementation in Phb1iΔIEC mice protected against ileitis and PC defects. Dysbiotic stool lysate decreased viability and increased apoptosis in Phb1-deficient enteroids that were prevented by butyrate supplementation. CONCLUSION Mitochondrial health in IECs cells is crucial for healthy gut microbiota composition and for epithelial defense when the microbiome becomes dysbiotic. Butyrate rescues IECs from insults caused by dysbiotic gut microbiota during mitochondrial dysfunction driven by PHB1 deletion.

Water-Reaching Platform for Longitudinal Assessment of Cortical Activity and Fine Motor Coordination Defects in a Huntington Disease Mouse Model.

Huntington disease (HD), caused by dominantly inherited expansions of a CAG repeat results in characteristic motor dysfunction. Although gross motor defects have been extensively characterized in multiple HD mouse models using tasks such as rotarod and beam walking, less is known about forelimb deficits. We develop a high-throughput alternating reward/nonreward water-reaching task and training protocol conducted daily over approximately two months to simultaneously monitor forelimb impairment and mesoscale cortical changes in GCaMP activity, comparing female zQ175 (HD) and wild-type (WT) littermate mice, starting at ∼5.5 months. Behavioral analysis of the water-reaching task reveals that HD mice, despite learning the water-reaching task as proficiently as wild-type mice, take longer to learn the alternating event sequence as evident by impulsive (noncued) reaches and initially display reduced cortical activity associated with successful reaches. At this age gross motor defects determined by tapered beam assessment were not apparent. Although wild-type mice displayed no significant changes in cortical activity and reaching trajectory throughout the testing period, HD mice exhibited an increase in cortical activity, especially in the secondary motor and retrosplenial cortices, over time, as well as longer and more variable reaching trajectories by approximately seven months. HD mice also experienced a progressive reduction in successful performance. Tapered beam and rotarod tests as well as reduced DARPP-32 expression (striatal medium spiny neuron marker) after water-reaching assessment confirmed HD pathology. The water-reaching task can be used to inform on a daily basis, HD and other movement disorder onset and manifestation, therapeutic intervention windows, and test drug efficacy.

Sleep-inducing effect of Rhynchophylline in EphA4 knockout mice.

We have recently demonstrated that the alkaloid rhynchophylline (RHY; purified from Uncaria plants) induces sleep and modifies electrocorticographic (ECoG) activity throughout the 24-h day in a vigilance state-dependent manner in wild-type mice. We here asked whether this alkaloid impacts wake/sleep variables in the absence of the cell adhesion protein EPHA4, via ECoG recording in EphA4 knockout (KO) mice submitted to the same RHY treatment contemporaneously to the wild-type mice (littermates). We uncover that RHY decreases time spent awake and increases time spent in slow wave sleep in EphA4 KO mice and alters the 24-h time course of ECoG activity during wakefulness and sleep states. These observations are similar to the reported effects of RHY in wild-type littermate animals, which strongly supports that RHY-driven sleep alterations are not dependent on the presence of EPHA4.

MUC13 Cell Surface Mucin Limits Salmonella Typhimurium Infection by Protecting the Mucosal Epithelial Barrier

MUC13 cell surface mucin is highlyexpressed on the mucosal surface throughout the intestine, yet its role against bacterial infection is unknown. We investigated how MUC13 impacts Salmonella typhimurium (S Tm) infection and elucidated its mechanisms of action. Muc13-/- and wild-type littermate mice were gavaged with 2 isogenic strains of S Tm after pre-conditioning with streptomycin. We assessed clinical parameters, cecal histology, local and systemic bacterial load, and proinflammatory cytokines after infection. Cecal enteroids and epithelial cell lines were used to evaluate the mechanism of MUC13 activity after infection. The interaction between bacterial SiiE and MUC13 was assessed by using siiE-deficient Salmonella. S Tm-infected Muc13-/- mice had increased disease activity, histologic damage, and higher local and systemic bacterial loads. Mechanistically, we found that S Tm binds to MUC13 through its giant SiiE adhesin and that MUC13 acts as a pathogen-binding decoy shed from the epithelial cell surface after pathogen engagement, limiting bacterial invasion. In addition, MUC13 reduces epithelial cell death and intestinal barrier breakdown by enhancing nuclear factor kappa B signaling during infection, independent of its decoy function. We show for the first time that MUC13 plays a critical role in antimicrobial defense against pathogenic S Tm at the intestinal mucosal surface by both acting as a releasable decoy limiting bacterial invasion and reducing pathogen-induced cell death. This further implicates the cell surface mucin family in mucosal defense from bacterial infection.

Delineating the Role of Toll-Like Receptors in Inflammatory Bowel Disease.

Toll-like receptors (TLRs) recognize altered gut microbiota triggering an immune response. These responses play a critical role in the pathogenesis and treatment of inflammatory bowel disease (IBD). IBD is characterized by inflammation of the intestinal tracts as in Crohn's disease and ulcerative colitis. However, one challenge in determining the role of a specific TLR in IBD and its underlying mechanism is disparity. Variance in age, gender, race, and ethnicity shows a dramatic difference in the disease incidence, severity, and response to treatment. Delineating the role of TLRs in IBD relies on both a knockout mouse and a disease model. Here, we describe a detailed protocol on how to use nearly identical genetic backgrounds of TLR wild-type and knockout littermate mice in a dextran sodium sulfate (DSS)-induced colitis model.

Multi‐omics analyses reveal unexpected effects of PLCg2 deficiency in the brain

AbstractBackground Plcg2 polymorphisms are associated with both reduced (P522R) and increased (M28L) risk of Alzheimer’s disease (AD). The normal function of phospholipase C‐gamma‐2 (PLCg2) protein is to catalyze the hydrolysis of the membrane phosphatidylinositol‐4,5‐bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol trisphosphate (IP3), which subsequently feed into downstream signaling pathways involving protein kinase C (PKC) activation, lipid homeostasis, and calcium regulation. In the brain, Plcg2 is primarily expressed by microglia. The Plcg2 P522R protective variant is a functional hypermorph, while it is predicted that M28L is a loss‐of‐function variant. The specific consequences of PLCg2 loss of function in the brain are unknown. We predicted that reduction of PLCg2 would lead to disrupted PIP2 and DAG metabolism, and that these changes would be associated with decreased PKC and calcium signaling resulting in changes in microglia phenotype.MethodsAt 3 months of age, PLCg2 knockout (KO), PLCg2 heterozygous, and wild‐type (WT) littermate mice were euthanized. We analyzed their brains using, shotgun lipidomics, proteomics, gene expression analysis by a Nanostring Glia Profiling panel, and immunohistochemistry (IHC).ResultsLipidomic analyses revealed expected but sex‐specific changes in total brain PIP2 and DAG content in KOs compared to WTs. Surprisingly, significant decreases in myelin‐specific lipids (e.g., cerebrosides) were identified in both sexes. Unexpectedly, Nanostring gene expression analysis revealed sex‐specific changes in myelin‐related genes, as well as subtle effects on microglia phenotype in KOs compared to WTs, suggestive of reduced microglia . Surprisingly, exploratory proteomics analyses revealed subtle changes in KO females compared to WTs, primarily related to amino acid metabolism. IHC for microglial markers confirmed subtle differences in microglia (Iba1 density and area) in KOs compared to WTs.ConclusionsOur data demonstrate that loss of PLCg2 in the brain generally leads to subtle differences in microglia phenotype in the absence of inflammatory stimuli (suggestive of reduced microglial reactivity) and a surprising potential role for PLCg2 in myelin and amino acid homeostasis that requires further investigation. Overall, our data indicate that loss of PLCg2 function has subtle effects on brain homeostasis that may underlie enhanced vulnerability to AD pathology via microglia and myelin dysfunction.

The DNA-Sensor AIM2 Promotes BCR-Activated B Cells in Chronic Graft-Versus-Host-Disease

Chronic graft-versus-host-disease (cGVHD) remains a common and poorly understood cause of late non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HCT). T cells and IgG-producing B cells mediate cGVHD development and perpetuation, but molecular mechanisms driving immune pathology require further study. We have shown that B cells from cGVHD patients are B Cell Receptor (BCR)-activated and primed for survival. In a mouse model of bone-marrow transplantation (BMT) with cGVHD manifestations, BCR signaling is promoted by alloantigens and BAFF (Jia et al., Blood 2021). BCR signaling is a viable therapeutic target in cGVHD, but other targetable pathways that promote pathological BCR-activated B cells remain largely unknown. Our single cell RNA-sequencing study of B cells from the peripheral blood (PB) of HCT patients identified 'Absent in Melanoma 2' (AIM2) as a prime candidate for further study (ASH abstract 2019, Poe et al., manuscript submitted). AIM2 was a signature gene in a BCR-activated subset after HCT, and AIM2 transcripts were significantly increased in a pre-germinal center (GC) B cell subset in cGVHD vs. no cGVHD patients (adjusted p=0.049). AIM2 incites inflammasome-mediated pyroptotic cell death in myeloid cells, but the function of AIM2 in B cells is largely unknown. AIM2 is expressed by CD27+ antigen (Ag)-experienced B cells in healthy individuals (HI) (Svensson et al., PlosOne 2017) and emerging evidence suggests that AIM2 promotes antibody production (Yang et al., Signal Transduct Target Ther 2021). We hypothesize that AIM2 promotes pathological BCR-activated B cells and anti-host IgG production in cGVHD. Using intracellular flow cytometry (IFC) and PB samples from HI (n=27), we first confirmed that the AIM2 protein is mainly expressed by CD27+ Ag-experienced B cells. Specifically, 88% and 99% of the IgD+ pre- and IgD- post-GC CD27+ B cells expressed AIM2, respectively. In HCT, we found a significantly higher proportion of AIM2+ cells in the circulating CD27+ IgD- post-GC B cells from cGVHD patients (n=7) vs. no cGVHD patients (n=5) (p=0.02). We also found significant overexpression of AIM2 transcripts in B cells from mice with cGVHD manifestations vs. no cGVHD mice (n=5/group, p=0.03, Figure 1A). To determine if Ag-activation together with other extrinsic factors typically encountered by B cells after HCT induced AIM2 expression by purified CD27- B cells from HI, we used IFC. The proportion of AIM2+ CD27- B cells increased after BCR +/- Toll-like receptor 7 or 9 stimulation only when IFNγ was present. Next, we studied the function of AIM2 in B cells. Unlike myeloid cells, CD27+ B cells from HI did not undergo pyroptotic cell death after stimulation with double stranded DNA. This lack of pyroptosis was not due to a deficiency in the inflammasome components and B cells had the capacity to undergo pyroptosis after exposure to the NLRP1/3 activator, nigericin. To assess effects of AIM2 on specific IgG production, we used AIM2 KO mice in a hapten-carrier immunization model (NP-KLH + alum i.p.). AIM2-KO mice had larger splenic GC vs. WT controls at day 8 post-immunization (p<0.0001). GC enlargement paradoxically associated with lower anti-NP IgG levels (p=0.03). Finally, having previously shown that BM+Spl recipients have a significantly higher proportion of GL7+ BCR-activated blood B cells (Jia et al., Blood 2021), we asked whether AIM2 promoted GL7+ B cells in cGVHD. To address this, we used AIM2-floxed mice, kindly supplied by Genentech, to breed AIM2fl/fl Mb1Cre/+ conditional KO mice (AIM2-CKO), deficient in AIM2 only in B cells. We then used these AIM2-CKO vs. AIM2fl/fl Mb1+/+ control littermates mice which are AIM2-sufficient as BMT donors. At day 24 post-BMT, recipients of BM+Spl from AIM2-CKO mice had a significantly lower percentage of mature (CD19+ CD93-) GL7+ B cells in PB vs. recipients of BM+Spl from the AIM2-sufficient littermates (n=10/group, p=0.004, Figure 1B). This was not due to baseline B cell differences in donor CKO mice. These results are being confirmed at later time points in ongoing, additional experiments. Our data show that AIM2 promotes IgG-producing B cells and circulating GL7+ B cells, suggesting that AIM2 plays a novel inflammasome-independent role in cGVHD B cell pathology. We are actively investigating how AIM2 effects alloantibody production and cGVHD development, so that therapeutic targets can be identified. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Deficiency of interleukin-6 receptor ameliorates PM2.5 exposure-induced pulmonary dysfunction and inflammation but not abnormalities in glucose homeostasis

BackgroundAmbient fine particulate matter (PM2.5) exposure increases local and systemic interleukin-6 (IL-6). However, the pathogenic role of IL-6 signalling following PM2.5 exposure, particularly in the development of pulmonary dysfunction and abnormal glucose homeostasis, has hardly been investigated. ResultsIn the study, IL-6 receptor (IL-6R)-deficient (IL-6R-/-) and wildtype littermate (IL-6R+/+) mice were exposed to concentrated ambient PM2.5 (CAP) or filtered air (FA), and their pulmonary and metabolic responses to these exposures were analyzed. Our results demonstrated that IL-6R deficiency markedly alleviated PM2.5 exposure-induced increases in lung inflammatory markers including the inflammation score of histological analysis, the number of macrophages in bronchoalveolar lavage fluid (BALF), and mRNA expressions of TNFα, IL-1β and IL-6 and abnormalities in lung function test. However, IL-6R deficiency did not reduce the hepatic insulin resistance nor systemic glucose intolerance and insulin resistance induced by PM2.5 exposure. ConclusionOur findings support the crucial role of IL-6 signalling in the development of pulmonary inflammation and dysfunction due to PM2.5 exposure but question the putative central role of pulmonary inflammation for the extra-pulmonary dysfunctions following PM2.5 exposure, providing a deep mechanistic insight into the pathogenesis caused by PM2.5 exposure.

Nintedanib Reduces Muscle Fibrosis and Improves Muscle Function of the Alpha-Sarcoglycan-Deficient Mice.

Sarcoglycanopathies are a group of recessive limb-girdle muscular dystrophies, characterized by progressive muscle weakness. Sarcoglycan deficiency produces instability of the sarcolemma during muscle contraction, leading to continuous muscle fiber injury eventually producing fiber loss and replacement by fibro-adipose tissue. Therapeutic strategies aiming to reduce fibro-adipose expansion could be effective in muscular dystrophies. We report the positive effect of nintedanib in a murine model of alpha-sarcoglycanopathy. We treated 14 Sgca-/- mice, six weeks old, with nintedanib 50 mg/kg every 12 h for 10 weeks and compared muscle function and histology with 14 Sgca-/- mice treated with vehicle and six wild-type littermate mice. Muscle function was assessed using a treadmill and grip strength. A cardiac evaluation was performed by echocardiography and histological study. Structural analysis of the muscles, including a detailed study of the fibrotic and inflammatory processes, was performed using conventional staining and immunofluorescence. In addition, proteomics and transcriptomics studies were carried out. Nintedanib was well tolerated by the animals treated, although we observed weight loss. Sgca-/- mice treated with nintedanib covered a longer distance on the treadmill, compared with non-treated Sgca-/- mice, and showed higher strength in the grip test. Moreover, nintedanib improved the muscle architecture of treated mice, reducing the degenerative area and the fibrotic reaction that was associated with a reversion of the cytokine expression profile. Nintedanib improved muscle function and muscle architecture by reducing muscle fibrosis and degeneration and reverting the chronic inflammatory environment suggesting that it could be a useful therapy for patients with alpha-sarcoglycanopathy.

Sleep Alterations in a Mouse Model of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder showing progressive neuronal loss in several brain areas and a broad spectrum of motor and non-motor symptoms, including ataxia and altered sleep. While sleep disturbances are known to play pathophysiologic roles in other neurodegenerative disorders, their impact on SCA3 is unknown. Using spectrographic measurements, we sought to quantitatively characterize sleep electroencephalography (EEG) in SCA3 transgenic mice with confirmed disease phenotype. We first measured motor phenotypes in 18–31-week-old homozygous SCA3 YACMJD84.2 mice and non-transgenic wild-type littermate mice during lights-on and lights-off periods. We next implanted electrodes to obtain 12-h (zeitgeber time 0-12) EEG recordings for three consecutive days when the mice were 26–36 weeks old. EEG-based spectroscopy showed that compared to wild-type littermates, SCA3 homozygous mice display: (i) increased duration of rapid-eye movement sleep (REM) and fragmentation in all sleep and wake states; (ii) higher beta power oscillations during REM and non-REM (NREM); and (iii) additional spectral power band alterations during REM and wake. Our data show that sleep architecture and EEG spectral power are dysregulated in homozygous SCA3 mice, indicating that common sleep-related etiologic factors may underlie mouse and human SCA3 phenotypes.