Wild-type Mice Research Articles

Knocking Out TAAR5: A Pathway to Enhanced Neurogenesis and Dopamine Signaling in the Striatum.

The member of trace-amine associated receptor family, TAAR5 receptor was suggested to recognize tertiary amines, mostly in the olfactory system; however, knocking out the receptor TAAR5 in mice showed an enhancing effect on adult neurogenesis and dopamine neurotransmission in the striatum. To estimate the role of the TAAR5, we performed gene expression profiling of striatal samples from TAAR5 knockout (KO) mice and their wild-type littermates. The higher expression of several genes involved in dopaminergic signaling and the downregulation of genes associated with gliogenesis were revealed in TAAR5-KO mice. Meanwhile, the upregulating effect of TAAR5 knockout on genes was associated with neurogenesis and synaptogenesis. The estimation of cell-type relative abundance through the deconvolution of RNA sequencing data demonstrated that TAAR5-KO striatum samples contain more D2 dopamine receptor-expressing medium spiny neurons but fewer astrocytes than wild-type mice. Our findings indicate that previously identified improvement in cognitive functions and motor coordination in TAAR5-KO mice may activate genes involved in neurogenesis, synaptogenesis, and synapse organization in the striatum. These data suggest that the pharmaceutical targeting of TAAR5 may improve striatum-dependent cognitive or motor functions. At the same time, a more detailed investigation of future TAAR5 antagonists' effect on glia development is necessary.

Dual calcium-voltage optical mapping of regional voltage and calcium signals in intact murine RyR2-R2474S hearts

Abnormal regional variations in electrical and calcium homeostasis properties have been implicated in catecholaminergic polymorphic ventricular tachycardias (CPVT) attributable to abnormal RyR2-mediated store Ca2+ release, but their underlying mechanism have not been well explored in intact hearts. MethodsWe performed in vivo and ex vivo studies including high throughput mapping of Ca2+ transients (CaT) and transmembrane voltage (Vm) in murine wild-type (WT) and heterozygous RyR2-R2474S/+ hearts, before and during isoprenaline (ISO) challenge. ResultsISO-challenged RyR2-R2474S/+ showed increased incidence of arrhythmia accompanied by abnormal Ca2+ transients compared to WT. CaT duration (CaTD) in the LV apex amongst regions studied both before and during ISO challenge in both WT and RyR2-R2474S/+ ventricles. RyR2-R2474S/+ ventricles showed prolonged CaTD, both before and during isoprenaline (ISO) challenge. Conversely, action potential durations (APD) were the same in WT and RyR2-R2474S/+ ventricles and identically reduced by ISO challenge. RyR2-R2474S/+ showed Vm-CaT latencies at time to half decay, but not rise time to peak, which were significantly prolonged compared to WT in all ventricular regions examined with ISO challenge. Following burst pacing, ventricular localized concordant alternans in CaT and APD were readily observed in RyR2-R2474S/+ but not in WT mice. Such CaT and APD alternans occurred mostly semiannually in specific regions of the ventricular pre-occurrence of VT. ConclusionThe pro-arrhythmic RyR2-R2474S/+ phenotype in intact hearts thus directly parallels delayed regional CaT recovery properties and alteration of Vm-CaT latencies. Studies suggest that discordant localized calcium alternans are mechanistically responsible for action potential duration alternans and occurrence of VT in RyR2-R2474S/+ mice.

Muscle type-specific effects of bilateral abobotulinumtoxinA injection on muscle growth and contractile function in spastic mice.

Intramuscular injection of botulinum neurotoxin type A (BoNT-A) is commonly used to improve or maintain the joint range of motion in young children with spasticity. However, the effectiveness of BoNT-A treatment is variable and movement limitations are recurrent. Here we show long-term effects of a single, bilateral abobotulinumtoxinA (aboBoNT-A) injection in the gastrocnemius medialis and soleus muscles of wild-type and spastic (B6.Cg-Glrbspa/J with a mutation in the glycine receptor) mice at a young age (6-7 days). Specifically, we evaluated the impact of aboBoNT-A-A on gait, physical performance, and spontaneous physical behavior, as well as on contractile force characteristics, morphology, and histological phenotype of soleus and gastrocnemius muscles by comparing their results to those of saline-injected controls up to 9 weeks after the injection. The detailed time course of the study specifies the timing of the aboBoNT-A injection at 1 week, the period of behavioral studies from 4-9 weeks, and the age of the mice (10 weeks) at the time of contractile force characteristics and histology assessments. In spastic mice, aboBoNT-A injection had a minor and very specific effect on physical performance, by only modestly increasing stride length as a function of age. aboBoNT-A injection caused a reduction in the force-generating capacity and a slightly smaller physiological cross-sectional area in gastrocnemius medialis, but not in soleus. Reduced physiological cross-sectional area in aboBoNT-A-injected muscles was due to a lower number of muscle fibers, rather than reduced muscle fiber cross-sectional area. The percentage of slow-type muscle fibers and mitochondrial succinate dehydrogenase activity were increased, which was associated with an improved muscle endurance capacity. In conclusion, aboBoNT-A injection reduced the number of muscle fibers, causing muscle hypertrophy in remaining fibers and a shift towards more oxidative fibers, resulting in an improved endurance capacity and gait. This study proposed potential cellular mechanisms for the therapeutic efficacy of aboBoNT-A in spasticity.

Elucidating the Metabolism of Chiral PCB95 in Wildtype and Transgenic Mouse Models with Altered Cytochrome P450 Enzymes Using Intestinal Content Screening.

Polychlorinated biphenyls (PCBs), such as 2,2',3,5',6-pentachlorobiphenyl (PCB95), are persistent organic pollutants associated with adverse health outcomes, including developmental neurotoxicity. PCB95 is a chiral neurotoxic PCB congener atropselectively metabolized to potentially neurotoxic metabolites in vivo. However, the metabolic pathways of most PCB congeners, including PCB95, remain unknown. To address this knowledge gap, we analyzed the intestinal contents of mice exposed to PCB95 to elucidate the PCB95 metabolism pathway and assess if genetic manipulation of hepatic drug-metabolizing enzymes affects PCB95 metabolism. Our study exposed male and female wildtype (WT), Cyp2abfgs-null (KO), and CYP2A6-transgenic/Cyp2abfgs-null (KI) mice orally to 1.0 mg/kg body weight of PCB95. Intestinal content was collected 24 h after PCB administration. aS-PCB95 was enriched in all intestinal content samples, irrespective of sex and genotype. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analyses identified 5 mono- (OH-PCB95) and 4 dihydroxylated PCB (diOH-PCB95) metabolites. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) identified 15 polar hydroxylated, methoxylated, and sulfated PCB95 metabolites, including 3 dechlorinated metabolites. A sex difference in the relative OH-PCB95 levels was observed only for KO in the LC-HRMS analysis. Genotype-dependent differences were observed for female, but not male, mice, with OH-PCB95 levels in female KO (FKO) mice tending to be lower than those in female WT (FWT) and KI (FKI) mice. Based on the GC-MS/MS analysis, these differences are due to the unknown PCB95 metabolites, X1-95 and Y1-95. These findings demonstrate that combining GC-MS/MS analyses and LC-HRMS subject screening of the intestinal content of PCB95-exposed mice can significantly advance our understanding of PCB95 metabolism in vivo.

FZD5 controls intestinal crypt homeostasis and colonic Wnt surrogate agonist response.

The rapidly regenerating intestinal epithelium requires crypt intestinal stem cells (ISCs). Wnt/β-catenin signaling maintains crypt homeostasis and Lgr5+ ISCs, and WNT ligands bind Frizzled receptors (FZD1-10). Identifying specific FZD(s) essential for intestinal homeostasis has been elusive; however, bioengineered antagonists blocking Wnt binding to FZD5 and FZD8 deplete the gut epithelium invivo, highlighting potential roles. Here, an epithelial-specific Fzd5 knockout (KO) elicited lethal pan-intestinal crypt and villus loss, whereas an Lgr5+ ISC-specific Fzd5 KO depleted Lgr5+ ISCs via premature differentiation and repressed Wnt target genes. Fzd5-null phenotypes were rescued by constitutive β-catenin activation invivo and in both mouse and human enteroids. KO of Fzd5, not Fzd8, in enteroids ablated responsiveness to dual-specificity FZD5/FZD8-selective Wnt surrogate agonists, which ameliorated DSS-induced colitis in wild-type and Fzd8 KO mice. Overall, FZD5 is a dominant and essential regulator of crypt homeostasis, Lgr5+ ISCs, and intestinal response to Wnt surrogate agonists, with implications for therapeutic mucosal repair.

CD4+ T cells drive corneal nerve damage but not epitheliopathy in an acute aqueous-deficient dry eye model

Dry eye disease (DED) is characterized by a dysfunctional tear film in which the corneal epithelium and its abundant nerves are affected by ocular desiccation and inflammation. Although adaptive immunity and specifically CD4+ T cells play a role in DED pathogenesis, the exact contribution of these cells to corneal epithelial and neural damage remains undetermined. To address this, we explored the progression of a surgical DED model in wild-type (WT) and T cell-deficient mice. We observed that adaptive immune-deficient mice developed all aspects of DED comparably to WT mice except for the absence of functional and morphological corneal nerve changes, nerve damage-associated transcriptomic signature in the trigeminal ganglia, and sustained tear cytokine levels. Adoptive transfer of CD4+ T cells from WT DED mice to T cell-deficient mice reproduced corneal nerve damage but not epitheliopathy. Conversely, T cell-deficient mice reconstituted solely with naïve CD4+ T cells developed corneal nerve impairment and epitheliopathy upon DED induction, thus replicating the WT DED phenotype. Collectively, our data show that while corneal neuropathy is driven by CD4+ T cells in DED, corneal epithelial damage develops independently of the adaptive immune response. These findings have implications for T cell-targeting therapies currently in use for DED.

Deficiency of lysophosphatidic acid receptor 3 decreases erythropoietin production in hypoxic mouse kidneys.

Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological functions through its receptors on the cell membrane. As one of the six LPA receptors, LPA receptor 3 (LPAR3) is highly expressed in mouse kidneys, but its physiological function in the kidney has been poorly explored. Wild-type (WT) and Lpar3-/- mice were used to investigate the renal physiological function of LPAR3 under hypoxia. The expression levels of LPA receptors in the kidneys of WT mice with or without exposure to hypoxia (8% O2) were detected by RT‒qPCR. RNA sequencing analysis was performed to identify differences in gene expression profiles between the hypoxic kidneys of WT and Lpar3-/- mice. The effects of LPAR3 deficiency and treatment with the LPAR1/3 inhibitor Ki16425 or the LPAR3 selective agonist 2S-OMPT on erythropoietin (EPO) production in the kidneys of hypoxic mice were determined by RT‒qPCR and ELISAs. The mechanism of LPAR3-mediated regulation of EPO expression was further studied in vivo with mouse models and in vitro with cultured human cells. LPAR3 is the major LPA receptor in mouse kidneys, and its expression is significantly upregulated under hypoxic conditions. RNA sequencing analysis revealed that, compared with WT mice, Lpar3-/- mice presented a significant decrease in hypoxia-induced EPO expression in the kidney, together with reduced plasma EPO levels and lower hematocrit and hemoglobin levels. Hypoxic renal EPO expression in WT mice was diminished by the administration of the LPAR1/3 inhibitor Ki16425 and increased by 2S-OMPT, a selective agonist of LPAR3. Hypoxia-induced HIF-2α accumulation in mouse kidneys was impaired by LPAR3 deficiency. Further studies revealed that the PI3K/Akt pathway participated in the regulation of HIF-2α accumulation and EPO expression by LPAR3 under hypoxic conditions. Our study revealed the role of LPAR3 in promoting the HIF-2α‒EPO axis in hypoxic mouse kidneys, suggesting that the LPA receptor may serve as a novel potential pharmaceutical target to regulate renal EPO production in hypoxia-related situations, such as chronic kidney disease and altitude disease.

Knockout of TMEM206 in mice associated with a loss of corneal transparency.

To investigate the role of transmembrane protein 206 (TMEM206) in corneal edema in mice by knockout the TMEM206 gene using CRISPR/Cas9 editing technology. TMEM206-knockout mice were generated using the CRISPR-Cas9 system. Variations in ophthalmic pathology were observed using slit lamp microscope and optical coherence tomography (OCT), intraocular pressure (IOP) was measured using a TonoLab Rebound Tonometer, and the ultrastructure of the corneal was observed using a transmission electron microscope. Corneal opacity was observed in 4/18 homozygous TMEM206-/- mice whereas a similar change was not observed in heterozygous TMEM206+/- mice and wild-type littermates. OCT examination showed that the mean central cornea thickness was 125±5.4 µm in 4 homozygous TMEM206-/- mice developed corneal edema and 115±1.2 µm in wild-type mice (t=3.468, P<0.05) at 43wk. The mean IOP was 12.08±0.07 mm Hg in four right eyes with corneal edema and 12.03±0.03 mm Hg in three normal left eyes (P>0.05). Transmission electron microscopy revealed a disruption in the organization of the collagen fibrils in the central part of the cornea in homozygous TMEM206-/- mice. TMEM206 is associated with corneal edema which caused organizational disruption of collagen fibrils in corneas of mice.

TET3 regulates terminal cell differentiation at the metabolic level

TET-family members play a critical role in cell fate commitment. Indeed, TET3 is essential to postnatal development due to yet unknown reasons. To define TET3 function in cell differentiation, we have profiled the intestinal epithelium at single-cell level from wild-type and Tet3 knockout mice. We have found that Tet3 is mostly expressed in differentiated enterocytes. In the absence of TET3, enterocytes exhibit an aberrant differentiation trajectory and do not acquire a physiological cell identity due to an impairment in oxidative phosphorylation, specifically due to an ATP synthase assembly deficiency. Moreover, spatial metabolomics analysis has revealed that Tet3 knockout enterocytes exhibit an unphysiological metabolic profile when compared with their wild-type counterparts. In contrast, no metabolic differences have been observed between both genotypes in the stem cell compartment where Tet3 is mainly not expressed. Collectively, our findings suggest a mechanism by which TET3 regulates mitochondrial function and, thus, terminal cell differentiation at the metabolic level.

Microsomal Prostaglandin E Synthase-1 Controls Colonic Prostaglandin E2 Production and Exerts a Protective Effect on Colitis Induced by Trinitrobenzene Sulfonic Acid in Mice.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an isozyme of the prostaglandin (PG) E synthase that acts downstream of cyclooxygenase and catalyzes the conversion of PGH2 to PGE2. The impact of genetic deletion of mPGES-1 on the development of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, a well-established model of inflammatory bowel disease (IBD), was investigated in this study. After administration of TNBS, mice deficient in mPGES-1 (mPGES-1-/- mice) showed more severe colitis than did wild-type (WT) mice. Histological examination revealed that mPGES-1-/- mice had markedly exacerbated symptoms of colitis. mPGES-1 expression was detectable in the colons of WT mice at both the mRNA and protein levels. Lack of mPGES-1 resulted in marked reduction of colonic PGE2 production. Our study also showed a significant increase in colonic expression of interleukin-17A (IL-17A), as well as interferon γ (IFNγ) and tumor necrosis factor α, during colitis in mPGES-1-/- mice compared with that in WT mice. Furthermore, loss of mPGES-1 increased the populations of IL-17A-producing T-helper (Th) 17 and IFNγ-producing Th1 cells in mesenteric lymph nodes. These results suggest that mPGES-1 is the main enzyme responsible for colonic PGE2 production and deficiency of mPGES-1 facilitates the development of colitis and T-cell-mediated immunity. mPGES-1 might, therefore, impact T-cell-related immune response associated with IBD.

Humanin-G Ameliorates Hemorrhage-Induced Acute Lung Injury in Mice Through AMPKα1-Dependent and -Independent Mechanisms.

Background/Objectives: The severity of acute lung injury is significantly impacted by age and sex in patients with hemorrhagic shock. AMP-activated protein kinase (AMPK) is a crucial regulator of energy metabolism but its activity declines with aging. Humanin is a mitochondrial peptide that exerts cytoprotective effects in response to oxidative stressors and is associated with longevity. Using a mouse model of hemorrhagic shock that mimics the clinical condition of adult patients, we investigated whether treatment with a humanin analog, humanin-G, mitigates lung injury and whether its mechanisms of action are dependent on the catalytic AMPKα1 subunit activation. Methods: Male and female AMPKα1 wild-type (WT) and knock-out (KO) mice (8-13 months old) were subjected to hemorrhagic shock by blood withdrawal, followed by resuscitation with shed blood and lactated Ringer's solution. The mice were treated with PEGylated humanin-G or vehicle and euthanized 3 h post-resuscitation. Results: Sex- and genotype-related differences were observed after hemorrhagic shock as lung neutrophil infiltration was more pronounced in the male AMPKα1 WT mice than the female WT mice; also, the male AMPKα1 KO mice experienced a significant decline in mean arterial blood pressure when compared to the male WT mice after resuscitation. The scores of histological lung injury were similarly elevated in all the male and female AMPKα1 WT and KO mice when compared to the control mice. At molecular analysis, acute lung injury was associated with the downregulation of AMPKα1/α2 catalytic subunits in the WT mice, whereas an increased activation of the signal transducer and activator of transcription-3 (STAT3) was observed in all the vehicle-treated groups. The in vivo administration of humanin-G ameliorated histological lung damage in all the groups of animals and ameliorated mean arterial blood pressure in the male AMPKα1 KO mice. The in vivo administration of humanin-G lowered lung neutrophil infiltration in the male and female AMPKα1 WT mice only but not in the KO mice. The beneficial results of humanin-G correlated with the lung cytosolic and nuclear activation of AMPKα in the male and female AMPKα1 WT groups, whereas STAT3 activation was not modified. Conclusions: In adult age, hemorrhage-induced acute lung injury manifests with sex-dependent characteristics. Humanin-G has therapeutic potential and the AMPKα1subunit is an important requisite for its inhibitory effects on lung leucosequestration, but not for the amelioration of lung alveolar structure or the hemodynamic effects of the peptide.

Liver knockout of MCU leads to greater dysregulation of lipid metabolism in MAFLD

Metabolic-associated fatty liver disease (MAFLD) is a common chronic condition that poses a significant threat to human health. Mitochondrial dysfunction, particularly involving the mitochondrial Ca2+ uniporter (MCU), plays a key role in its pathogenesis. This study aimed to investigate the impact of the MCU gene on hepatic lipid metabolism in mice fed a high-fat diet. Using MCU knockout and wild-type mice, subjected to either a high-fat or normal diet for 14 weeks, we observed notable Steatosis and liver weight gain in MCU-deficient mice. Liver function markers, serum triglycerides, very low-density lipoprotein (VLDL) levels, and ApoB protein expression were all significantly elevated. Mechanistic studies revealed that MCU deletion led to mitochondrial dysfunction, increased oxidative stress. These findings highlight the critical role of the MCU gene in maintaining hepatic lipid balance and suggest its potential as a therapeutic target for managing nonalcoholic fatty liver disease.

GRK2 mediates cisplatin-induced acute liver injury via the modulation of NOX4

BackgroundThe present study investigated the function of G protein-coupled receptor kinase 2 (GRK2) in acute liver injury (ALI) by cisplatin, and investigated the protective effect of pharmacological inhibition of GRK2.MethodsALI models were generated in global adult hemizygous (ALI-Grk2±) mice and wild-type (WT) mice. Liver biochemistry parameters and histopathology were used to evaluate the severity of ALI and the protective effect of pharmacological inhibition of GRK2. GRK2-siRNA was used to knock down the expression of GRK2 in AML12 cells in vitro.ResultsALI model mice exhibited increased blood levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and abnormal liver pathology accompanied by imbalanced L-glutathione (GSH) levels. Cisplatin administration upregulated GKR2, p-GRK2 and NADPH oxidase 4 (NOX4) expression in the liver tissues of ALI model mice. Compared to WT mice injected with cisplatin, Grk2± mice that received cisplatin showed significant improvements in liver function and pathological performance, decreased NOX4 levels, reduced endoplasmic reticulum (ER) stress, and diminished liver cell apoptosis. In vitro, the transfection of AML12 cells with siRNA significantly reduced NOX4 expression and inhibited cisplatin-induced reactive oxygen species production, ER stress (increased levels of GRP94, GRP78, p-elF2α and CHOP) and apoptotic death. Moreover, pharmacological treatment with drugs that inhibit GRK2 (CP-25 or paroxetine) significantly ameliorated cisplatin-induced ALI by improving liver pathological manifestations, inhibiting oxidative stress and ER stress, and reducing liver cell apoptosis. Similar results were observed in vitro.ConclusionsGRK2 mediates the development of cisplatin-induced ALI by modulating NOX4 and ER stress. Pharmacological inhibition of GRK2 with CP-25 or paroxetine effectively alleviated ALI. GRK2 can be used as a potential target for the prevention and treatment of liver injury.Graphical

Organophosphate Insecticide Malathion Induces Alzheimer's Disease-Like Cognitive Impairment in Mice: Evidence of the Microbiota-Gut-Brain Axis.

Evidence suggests that exposure to organophosphate pesticides increases the risk of neurodegenerative diseases, but the mechanisms remain unclear. This study investigated the effects of malathion on Alzheimer's disease (AD)-like symptoms at environmentally relevant concentrations using wild-type (WT) and APP/PS1 transgenic mouse models. Results showed that malathion exposure induced AD-like cognitive impairment, amyloid-β (Aβ) accumulation, and neuroinflammation in WT mice, with worsened symptoms in APP/PS1 mice. Mechanistic studies revealed that malathion induced AD-like gut microbiota dysbiosis (reduced Lactobacillus and Akkermansia, and increased Dubosiella), causing gut barrier impairment and tryptophan metabolism disruptions. This resulted in a significant increase in indole derivatives and activation of the colonic aryl hydrocarbon receptor (AhR), promoting the kynurenine (KYN) pathway while inhibiting the serotonin (5-HT) pathway. Increased neurotoxic KYN metabolites (3-hydroxykynurenine and quinolinic acid) triggered gut and systemic inflammation, upregulating hippocampal IL-6 and IL-1β mRNA levels and thereby causing neuroinflammation. Gut tryptophan metabolism disruptions caused hippocampal neurotransmitter imbalances, reducing the levels of 5-HT and its derivatives. These effects promoted AD progression in both WT and APP/PS1 mice. This study highlights the crucial role of the microbiota-gut-brain axis in AD-like cognitive impairment induced by malathion exposure, providing insights into the neurodegenerative disease risks posed by organophosphate pesticides.

The Fundamental Neurobiological Mechanism of Oxidative Stress-Related 4E-BP2 Protein Deamidation.

Memory impairment is caused by the absence of the 4E-BP2 protein in the brain. This protein undergoes deamidation spontaneously in the neurons. 4E-BP2 deamidation significantly alters protein synthesis in the neurons and affects the balance of protein production required for a healthy nervous system. Any imbalance in protein production in the nervous system causes neurodegenerative diseases. Discovering what causes 4E-BP2 deamidation will make it possible to control this balance of protein production and develop effective treatments against neurodegenerative diseases such as Alzheimer's and Parkinson's. The purpose of this work is to discover the neurobiological mechanism that causes the deamidation reaction in the 4E-BP2 protein by performing immunoblotting in the retinal ganglia, the optic nerve, the dorsal root ganglia, the sciatic nerve, and the whole brain, extracted via dissection from 2-month-old, Wild-type male mice. The results show that axons and their unique properties cause neuron-specific 4E-BP2 deamidation in the nervous system, confirming conclusively that axons are the critical factors behind the fundamental neurobiological mechanism of 4E-BP2 protein deamidation.

Bone phenotyping of murine hemochromatosis models with deficiencies of Hjv, Alk2, or Alk3: The influence of sex and the bone compartment.

Osteopenia is frequently observed in patients with iron overload, especially in those with HFE-dependent hereditary hemochromatosis (HH). Interestingly, not all mouse models of HH show bone loss, suggesting that iron overload alone may not suffice to induce bone loss. In this study, the bone phenotypes of Hjv-/- and hepatocyte-specific Alk2- and Alk3-deficient mice as additional mouse models of HH were investigated to further clarify, how high iron levels lead to bone loss and which signaling mechanisms are operational. Neither male nor female 12-week-old Hjv-/- mice had an altered trabecular or cortical bone mass or bone turnover, despite severe iron overload. Male 12-month-old Hjv-/- mice even presented with a higher femoral trabecular bone volume compared to wildtype mice. Similarly, female mice with hepatocyte-specific Alk2 or Alk3 deficiency did not show an altered bone phenotype at 3, 6, and 12 months of age. Male hepatocyte-specific Alk3-deficient mice also had a normal trabecular bone mass at all ages analyzed, despite showing increased bone resorption and decreased bone formation parameters. Interestingly, hepatocyte-specific Alk2-deficient mice showed reduced femoral trabecular bone at 6 months of age due to suppressed bone formation. Cortical thickness at the femur was reduced in both, 6-month-old male hepatocyte-specific Alk2- and Alk3-deficient mice. Raising hepatocyte-specific Alk2-deficient male mice on an iron-deficient diet rescued the bone phenotype. Taken together, despite iron overload, trabecular bone microarchitecture was not altered in mice deficient of Hjv or Alk3. Only male hepatocyte-specific Alk2-deficient mice showed site-specific lower trabecular and cortical bone mass at the femur, which was dependent on iron. Thus, bone loss does not correlate with the extent of iron overload in these mouse models, but may relate to the amount of iron-loaded macrophages, as precursors of osteoclasts, in the bone marrow.

The importance of mast cell histamine secretion in IgG-mediated systemic anaphylaxis.

IgG can mediate murine and human systemic anaphylaxis (SA). The roles of mast cells (MCs) and histamine in IgG-mediated anaphylaxis are controversial for mice and have not been studied in vivo for humans. We now investigate these issues. Actively or passively sensitized wild-type and immune-deficient mice were induced to develop anaphylaxis by i.v. antigen challenge. Anaphylaxis was characterized by evaluating hypothermia, hypomobility, histamine, and mast cell protease responses. In contrast to our previous results with protein-immunized mice from a conventional colony, IgG-mediated passive SA in our SPF colony mice depended considerably on histamine produced by connective tissue MCs (CTMCs) in response to FcγRIII crosslinking. This was found for C57BL/6 and young male and female BALB/c mice, including BALB/c mice freshly arrived from 3 vendors. IgG-mediated anaphylaxis was less histamine-dependent in old than young mice. Although both mucosal MC (MMC) and CTMC responses were severely depleted in c-kit-deficient mice, MMC responses depended considerably more than CTMC responses on c-kit for maintenance. In immunologically naïve mice, FcγRIII crosslinking strongly activated a subset of CTMCs, but had little ability to activate MMCs. In vivo LPS + poly I.C treatment decreased histamine-dependence of IgG-mediated anaphylaxis while a strong Th2 immune response increased FcγRIII crosslinking-induced MMC activation. IgG-mediated activation of human MCs in reconstituted immunodeficient mice induced histamine-dependent anaphylaxis. IgG-dependent SA can be mediated largely by histamine released by mouse CTMCs and human MCs; histamine dependence is influenced by mouse age, sex, immune and infectious history, and the anaphylaxis model studied.

Chemogenetic stimulation of phrenic motor output and diaphragm activity.

Impaired respiratory motor output contributes to morbidity and mortality in many neurodegenerative diseases and neurologic injuries. We investigated if expressing designer receptors exclusively activated by designer drugs (DREADDs) in the mid-cervical spinal cord could effectively stimulate phrenic motor output to increase diaphragm activation. Two primary questions were addressed: 1) does effective DREADD-mediated diaphragm activation require focal expression in phrenic motoneurons (vs. non-specific mid-cervical expression), and 2) can this method produce a sustained increase in inspiratory tidal volume? Wild type (C57/bl6) and ChAT-Cre mice received bilateral intraspinal (C4) injections of an adeno-associated virus (AAV) encoding the hM3D(Gq) excitatory DREADD. In wild-type mice, this produced non-specific DREADD expression throughout the mid-cervical ventral horn. In ChAT-Cre mice, a Cre-dependent viral construct was used to drive neuronal DREADD expression in the C4 ventral horn, targeting phrenic motoneurons. Diaphragm EMG was recorded in isoflurane-anesthetized spontaneously breathing mice at 4-9 weeks post-AAV delivery. The DREADD ligand JHU37160 (J60) caused a bilateral, sustained (>1 hour) increase in inspiratory EMG bursting in both groups; the relative increase was greater in ChAT-Cre mice. Additional experiments in ChAT-Cre rats were conducted to determine if spinal DREADD activation could increase inspiratory tidal volume (VT) during spontaneous breathing, assessed using whole-body plethysmography without anesthesia. Three-to-four months after intraspinal (C4) injection of AAV driving Cre-dependent hM3D(Gq) expression, intravenous J60 resulted in a sustained (>30 min) increase in VT. Subsequently, phrenic nerve recordings performed under urethane anesthesia confirmed that J60 evoked a > 200% increase in inspiratory output. We conclude that targeting mid-cervical spinal DREADD expression to the phrenic motoneuron pool enables ligand-induced, sustained increases in phrenic motor output and VT. Further development of this technology may enable application to clinical conditions associated with impaired diaphragm activation and hypoventilation.

CD47 and thrombospondin-1 contribute to immune evasion by Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative anaerobic bacterium linked to periodontal disease. Remarkably, P. gingivalis thrives in an inflamed environment rich in activated neutrophils. Toll-like receptor 2 (TLR2) recognition is required for P. gingivalis to evade innate immune killing; however, the mechanisms through which P. gingivalis uncouples host inflammation from bactericidal activity are only partially known. Since integrin activation and alternative signaling are implicated in P. gingivalis TLR2-mediated immune escape, we explored the role of CD47, a widely expressed integrin-associated protein known to suppress phagocytosis and implicated as an interacting partner with other innate immune receptors. We found that CD47 associates with TLR2, and blocking CD47 leads to decreased intracellular P. gingivalis survival in macrophages in a manner dependent on the bacterial major fimbria. In vivo, CD47 knock-out mice cleared P. gingivalis more efficiently than wild-type mice. Next, we found increased expression and secretion of the CD47 ligand thrombospondin-1 (TSP-1) following P. gingivalis infection. Secreted TSP-1 broadly protected P. gingivalis and other periodontitis-associated bacterial species from neutrophil bactericidal activity. Therefore, CD47-TLR2 cosignaling in response to P. gingivalis induces TSP-1 that in turn suppresses neutrophil activity, an effect that can explain how species such as P. gingivalis survive in an inflamed environment and cause dysbiosis.

Mitochondria-Targeted DNA Repair Glycosylase hOGG1 Protects Against HFD-Induced Liver Oxidative Mitochondrial DNA Damage and Insulin Resistance in OGG1-Deficient Mice.

8-oxoguanine DNA glycosylase-1 (OGG1) is a DNA glycosylase mediating the first step in base excision repair which removes 7,8-dihydro-8-oxoguanine (8-oxoG) and repairs oxidized nuclear and mitochondrial DNA. Previous studies showed that OGG1 deficiency results in an increased susceptibility to high-fat diet (HFD)-induced obesity and metabolic dysfunction in mice, suggesting a crucial role of OGG1 in metabolism. However, the tissue-specific mechanisms of how OGG1 deficiency leads to insulin resistance is unknown. Thus, in the current study, we used a hyperinsulinemic-euglycemic clamp to evaluate in-depth glucose metabolism in male wild-type (WT) mice and Ogg1-/- (Ogg1-KO) mice fed an HFD. Ogg1-KO mice fed HFD were more obese, with significantly lower hepatic insulin action compared to WT/HFD mice. Targeting human OGG1 to mitochondria protected against HFD-induced obesity, insulin resistance, oxidative mitochondrial DNA damage in the liver and showed decreased expression of liver gluconeogenic genes in Ogg1-KO mice, suggesting a putative protective mechanism. Additionally, several subunits of oxidative phosphorylation protein levels were noticeably increased in Ogg1-KO/Tg compared to Ogg1-KO mice fed an HFD which was associated with improved insulin signaling. Our findings demonstrate the crucial role of mitochondrial hOGG1 in HFD-induced insulin resistance and propose several protective mechanisms which can further direct the development of therapeutic treatment.