Normal hematopoiesis and inflammatory responses despite discrete signaling defects in Galpha15 knockout mice

Galpha15 activates phospholipase Cbeta in response to the greatest variety of agonist-stimulated heptahelical receptors among the four Gq class G-protein alpha subunits expressed in mammals. Galpha15 is primarily expressed in hematopoietic cells in fetal and adult mice. We disrupted the Galpha15 gene by homologous recombination in embryonic stem cells to identify its biological functions. Surprisingly, hematopoiesis was normal in Galpha15(-/-) mice, Galpha15(-/-) Galphaq(-/-) double-knockout mice (which express only Galpha11 in most hematopoietic cells), and Galpha11(-/-) mice, suggesting functional redundancy in Gq class signaling. Inflammatory challenges, including thioglycolate-induced peritonitis and infection with Trichinella spiralis, stimulated similar responses in Galpha15(-/-) adults and wild-type siblings. Agonist-stimulated Ca(2+) release from intracellular stores was assayed to identify signaling defects in primary cultures of thioglycolate-elicited macrophages isolated from Galpha15(-/-) mice. C5a-stimulated phosphoinositide accumulation and Ca(2+) release was significantly reduced in Galpha15(-/-) macrophages. Ca(2+) signaling was abolished only in mutant cells pretreated with pertussis toxin, suggesting that the C5a receptor couples to both Galpha15 and Galphai in vivo. Signaling evoked by other receptors coupled by Gq class alpha subunits appeared normal in Galpha15(-/-) macrophages. Despite discrete signaling defects, compensation by coexpressed Gq and/or Gi class alpha subunits may suppress abnormalities in Galpha15-deficient mice.     

Renal defects associated with improper polarization of the CRB and DLG polarity complexes in MALS-3 knockout mice

Kidney development and physiology require polarization of epithelia that line renal tubules. Genetic studies show that polarization of invertebrate epithelia requires the crumbs, partition-defective-3, and discs large complexes. These evolutionarily conserved protein complexes occur in mammalian kidney; however, their role in renal development remains poorly defined. Here, we find that mice lacking the small PDZ protein mammalian LIN-7c (MALS-3) have hypomorphic, cystic, and fibrotic kidneys. Proteomic analysis defines MALS-3 as the only known core component of both the crumbs and discs large cell polarity complexes. MALS-3 mediates stable assembly of the crumbs tight junction complex and the discs large basolateral complex, and these complexes are disrupted in renal epithelia from MALS-3 knockout mice. Interestingly, MALS-3 controls apico-basal polarity preferentially in epithelia derived from metanephric mesenchyme, and defects in kidney architecture owe solely to MALS expression in these epithelia. These studies demonstrate that defects in epithelial cell polarization can cause cystic and fibrotic renal disease.     

Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis

Triple helix formation of procollagen after the assembly of three alpha-chains at the C-propeptide is a prerequisite for refined structures such as fibers and meshworks. Hsp47 is an ER-resident stress inducible glycoprotein that specifically and transiently binds to newly synthesized procollagens. However, the real function of Hsp47 in collagen biosynthesis has not been elucidated in vitro or in vivo. Here, we describe the establishment of Hsp47 knockout mice that are severely deficient in the mature, propeptide-processed form of alpha1(I) collagen and fibril structures in mesenchymal tissues. The molecular form of type IV collagen was also affected, and basement membranes were discontinuously disrupted in the homozygotes. The homozygous mice did not survive beyond 11.5 days postcoitus (dpc), and displayed abnormally orientated epithelial tissues and ruptured blood vessels. When triple helix formation of type I collagen secreted from cultured cells was monitored by protease digestion, the collagens of Hsp47+/+ and Hsp47+/- cells were resistant, but those of Hsp47-/- cells were sensitive. These results indicate for the first time that type I collagen is unable to form a rigid triple-helical structure without the assistance of molecular chaperone Hsp47, and that mice require Hsp47 for normal development.     

Immunopathologic effects associated with Sarcocystis neurona-infected interferon-gamma knockout mice

Interferon-gamma knockout (IFN-gamma KO) mice were infected with Sarcocystis neurona merozoites to characterize the immunopathology associated with infection. By day 14 postinfection (PI), mice developed splenomegaly and lymphadenopathy, characterized by marked lymphoid hyperplasia with increased numbers of germinal centers. Additional histopathologic changes included increased extramedullary hematopoiesis, multifocal mixed inflammatory infiltrates in the liver, perivascular infiltrate of the liver and lung, and interstitial pneumonia. The total number of B-cell splenocytes (P < 0.05) and the percentage of B-cells increased on day 14 PI in the spleen and on day 28 PI in the lymph nodes (P < 0.05). By day 28 PI, the number of B-cell splenocytes decreased significantly. A non-subset-specific decrease in percentages of CD4 lymphocytes throughout all lymphoid organs was observed on day 14 PI. However, total CD4 and CD44/CD4 splenocytes increased significantly by day 28 PI. Early-activation CD8 lymphocytes were reduced in the blood and spleen, whereas memory CD8 lymphocyte percentages and total numbers were significantly increased. On the basis of the results, we propose that S. neurona-infected IFN-gamma KO mice are immunocompromised and unable to clear the infection. Thus, they develop B-cell exhaustion and a delayed, but sustained, increased number of memory CD4 and CD8 lymphocytes due to chronic antigen stimulation.     

Neural and orofacial defects in Folp1 knockout mice [corrected

BACKGROUND

Folic acid is essential for the development of the nervous system and other associated structures. Mice deficient in the folic acid‐binding protein one (Folbp1) gene display multiple developmental abnormalities, including neural and craniofacial defects. To better understand potential interactions between Folbp1 gene and selected genes involved in neural and craniofacial morphogenesis, we evaluated the expression patterns of a panel of crucial differentiation markers (Pax‐3, En‐2, Hox‐a1, Shh, Bmp‐4, Wnt‐1, and Pax‐1).

METHODS

Folbp1 mice were supplemented with low dosages of folinic acid to rescue nullizygotes from dying in utero before gestational day 10. The gene marker analyses were carried out by in situ hybridization.

RESULTS

In nullizygote embryos with open cranial neural tube defects, the downregulation of Pax‐3 and En‐2 in the impaired midbrain, along with an observed upregulation of the ventralizing marker Shh in the expanded floor plate, suggested an important regulatory interaction among these three genes. Moreover, the nullizygotes also exhibit craniofacial abnormalities, such as cleft lip and palate. Pax‐3 signals in the impaired medial nasal primordia were significantly increased, whereas Pax‐1 showed no expression in the undeveloped lateral nasal processes. Although Shh was downregulated, Bmp‐4 was strongly expressed in the medial and lateral nasal processes, highlighting the antagonistic activities of these molecules.

CONCLUSIONS

Impairment of Folbp1 gene function adversely impacts the expression of several critical signaling molecules. Mis‐expression of these molecules, perhaps mediated by Shh, may potentially contribute to the observed failure of neural tube closure and the development of craniofacial defects in the mutant mice. Birth Defects Research (Part A) 67:209–218, 2003. © 2003 Wiley‐Liss, Inc.

     

Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice

In vitro studies indicate a role for the LIM kinase family in the regulation of cofilin phosphorylation and actin dynamics. In addition, abnormal expression of LIMK-1 is associated with Williams syndrome, a mental disorder with profound deficits in visuospatial cognition. However, the in vivo function of this family of kinases remains elusive. Using LIMK-1 knockout mice, we demonstrate a significant role for LIMK-1 in vivo in regulating cofilin and the actin cytoskeleton. Furthermore, we show that the knockout mice exhibited significant abnormalities in spine morphology and in synaptic function, including enhanced hippocampal long-term potentiation. The knockout mice also showed altered fear responses and spatial learning. These results indicate that LIMK-1 plays a critical role in dendritic spine morphogenesis and brain function.     

Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice

SMAD4 is a central component of the TGFbeta superfamily signaling pathway. Within the ovary, TGFbeta-related proteins play crucial roles in controlling granulosa cell growth, differentiation, and steroidogenesis. To study the in vivo roles of SMAD4 during follicle development, we generated an ovarian conditional knockout of Smad4 using the cre/loxP recombination system. Smad4 ovarian-specific knockout mice are subfertile with decreasing fertility over time and multiple defects in folliculogenesis. Regulation of steroidogenesis is disrupted in the Smad4 conditional knockout, leading to increased levels of serum progesterone. In addition, severe cumulus cell defects are present both in vivo and when assayed in vitro. These findings demonstrate that disrupting signaling through SMAD4 in the ovarian granulosa cells leads to premature luteinization of granulosa cells and eventually premature ovarian failure, thereby demonstrating key in vivo roles of TGFbeta superfamily signaling in the timing of granulosa cell differentiation.     

Increased neuronal excitability, synaptic plasticity, and learning in aged Kvbeta1.1 knockout mice

BACKGROUND: Advancing age is typically accompanied by deficits in learning and memory. These deficits occur independently of overt pathology and are often considered to be a part of "normal aging." At the neuronal level, normal aging is known to be associated with numerous cellular and molecular changes, which include a pronounced decrease in neuronal excitability and an altered induction in the threshold for synaptic plasticity. Because both of these mechanisms (neuronal excitability and synaptic plasticity) have been implicated as putative cellular substrates for learning and memory, it is reasonable to propose that age-related changes in these mechanisms may contribute to the general cognitive decline that occurs during aging. RESULTS: To further investigate the relationship between aging, learning and memory, neuronal excitability, and synaptic plasticity, we have carried out experiments with aged mice that lack the auxiliary potassium channel subunit Kvbeta1.1. In aged mice, the deletion of the auxiliary potassium channel subunit Kvbeta1.1 resulted in increased neuronal excitability, as measured by a decrease in the post-burst afterhyperpolarization. In addition, long-term potentiation (LTP) was more readily induced in aged Kvbeta1.1 knockout mice. Finally, the aged Kvbeta1.1 mutants outperformed age-matched controls in the hidden-platform version of the Morris water maze. Interestingly, the enhancements in excitability and learning were both sensitive to genetic background: The enhanced learning was only observed in a genetic background in which the mutants exhibited increased neuronal excitability. CONCLUSIONS: Neuronal excitability is an important determinant of both synaptic plasticity and learning in aged subjects.     

Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.     

Longitudinal in vivo developmental changes of metabolites in the hippocampus of Fmr1 knockout mice

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is studied in the Fmr1 knockout (KO) mouse, which models both the anatomical and behavioral changes observed in FXS patients. In vitro studies have shown many alterations in synaptic plasticity and increased density of immature dendritic spines in the hippocampus, a region involved in learning and memory. In this study, magnetic resonance imaging (MRI) and ¹H magnetic resonance spectroscopy (MRS) were used to determine in vivo longitudinal changes in volume and metabolites in the hippocampus during the critical period of early myelination and synaptogenesis at post‐natal days (PND) 18, 21, and 30 in Fmr1 KO mice compared with wild‐type (WT) controls. MRI demonstrated an increase in volume of the hippocampus in the Fmr1 KO mouse compared with controls. MRS revealed significant developmental changes in the ratios of hippocampal metabolites N‐acetylaspartate (NAA), myo‐inositol (Ins), and taurine to total creatine (tCr) in Fmr1 KO mice compared with WT controls. Ins was decreased at PND 30, and taurine was increased at all ages studied in Fmr1 KO mice compared with controls. An imbalance of brain metabolites in the hippocampus of Fmr1 KO mice during the critical developmental period of synaptogenesis and early myelination could have long‐lasting effects that adversely affect brain development and contribute to ongoing alterations in brain function.     

Lung defects in neonatal and adult stromal-derived factor–1 conditional knockout mice

Stromal-derived factor (SDF)-1/CXCL12 is a cytokine that is involved in organogenesis, hematopoiesis, chemoattraction, and wound healing. An SDF-1 knockout mouse (SDF-1^-/-) has provided important insights into the role of SDF-1 in fetal development. Because the SDF-1 knockout is lethal in the perinatal period, we have created a conditional SDF-1 knockout mouse. In the present study, we induced conditionally knocked out SDF-1 in neonatal mice and found that lung development was compromised; neonatal lungs showed increased alveolar airspace and abnormal ultrastructure. Conditional knockout of SDF-1 in adult mice resulted in an emphysemic morphology, with increased alveolar airspace and thickened alveolar septa. Fluorescence angiography showed pulmonary vessel hyperdilation. To determine whether the hyperdilation involved nitric oxide, we inhibited endothelial nitric oxide synthase (eNOS) with N (G)-nitro-L- arginine methyl ester. This resulted in the inhibition of pulmonary vessel hyperdilation. Western blot results showed increased phosphorylation of eNOS in our induced SDF-1 knockout mice, indicating that eNOS is normally repressed in the presence of SDF-1, and that activation of eNOS contributes to pulmonary pathology. Thus, a conditional knockout mouse has been successsfully created for SDF-1; initial characterization indicates that SDF-1 is intimately involved in lung development and physiology.     

Increased anxiety-like behavior and enhanced synaptic efficacy in the amygdala of GluR5 knockout mice

GABAergic transmission in the amygdala modulates the expression of anxiety. Understanding the interplay between GABAergic transmission and excitatory circuits in the amygdala is, therefore, critical for understanding the neurobiological basis of anxiety. Here, we used a multi-disciplinary approach to demonstrate that GluR5-containing kainate receptors regulate local inhibitory circuits, modulate the excitatory transmission from the basolateral amygdala to the central amygdala, and control behavioral anxiety. Genetic deletion of GluR5 or local injection of a GluR5 antagonist into the basolateral amygdala increases anxiety-like behavior. Activation of GluR5 selectively depolarized inhibitory neurons, thereby increasing GABA release and contributing to tonic GABA current in the basolateral amygdala. The enhanced GABAergic transmission leads to reduced excitatory inputs in the central amygdala. Our results suggest that GluR5 is a key regulator of inhibitory circuits in the amygdala and highlight the potential use of GluR5-specific drugs in the treatment of pathological anxiety.     

Oxytocin knockout mice demonstrate enhanced intake of sweet and nonsweet carbohydrate solutions

Oxytocin knockout (OT KO) mice display enhanced intake of nutritive and nonnutritive sweet solutions (i.e., sucrose and saccharin) compared with wild-type (WT) mice of the same C57BL/6 background strain. The present study further investigated the differential behavioral response of OT KO and WT mice to sucrose solutions and also examined intake preferences of OT KO and WT mice for palatable but nonsweet isocaloric solutions of carbohydrate and fat. A progressive ratio operant licking procedure demonstrated that OT KO and WT mice display a similar motivational drive to consume 10% sucrose. A series of two-bottle intake tests revealed that OT KO mice consume significantly larger amounts of both sweet and nonsweet carbohydrate solutions (i.e., sucrose, Polycose, and cornstarch) compared with WT cohorts. Intake pattern analyses revealed that OT KO mice overconsume carbohydrate solutions by initiating more drinking bouts compared with WT mice; bout sizes did not differ between the genotypes. In contrast, OT KO and WT mice did not differ in their intake of Intralipid, a palatable soybean oil emulsion. These findings indicate that the absence of OT in mice does not affect their appetitive drive to consume palatable sucrose solutions. Instead, the absence of OT may increase daily intake of palatable sweet and nonsweet solutions of carbohydrate (but not fat) by selectively blunting or masking processes that contribute to postingestive satiety.     

Accelerated resolution of AA amyloid in heparanase knockout mice is associated with matrix metalloproteases

AA-amyloidosis is a disease characterized by abnormal deposition of serum A amyloid (SAA) peptide along with other components in various organs. The disease is a complication of inflammatory conditions that cause persistent high levels of the acute phase reactant SAA in plasma. In experimental animal models, the deposited amyloid is resolved when the inflammation is stopped, suggesting that there is an efficient clearance mechanism for the amyloid. As heparan sulfate (HS) is one of the major components in the amyloid, its metabolism is expected to affect the pathology of AA amyloidosis. In this study, we investigated the effect of heparanase, a HS degradation enzyme, in resolution of the AA amyloid. The transgenic mice deficient in heparanase (Hpa-KO) produced a similar level of SAA in plasma as the wildtype control (Ctr) mice upon induction by injection of AEF (amyloid enhancing factor) and inflammatory stimuli. The induction resulted in formation of SAA amyloid 7-days post treatment in the spleen that displayed a comparable degree of amyloid load in both groups. The amyloid became significantly less in the Hpa-KO spleen than in the Ctr spleen 10-days post treatment, and was completely resolved in the Hpa-KO spleen on day 21 post induction, while a substantial amount was still detected in the Ctr spleen. The rapid clearance of the amyloid in the Hpa-KO mice can be ascribed to upregulated matrix metalloproteases (MMPs) that are believed to contribute to degradation of the protein components in the AA amyloid. The results indicate that both heparanase and MMPs play important parts in the pathological process of AA amyloidosis.     

Invariant NKT cell defects in vitamin D receptor knockout mice prevents experimental lung inflammation

Vitamin D receptor (VDR) deficiency (knockout [KO]) results in a failure of mice to generate an airway hyperreactivity (AHR) response on both the BALB/c and C57BL/6 background. The cause of the failed AHR response is the defective population of invariant NKT (iNKT) cells in the VDR KO mice because wild-type (WT) iNKT cells rescued the AHR response. VDR KO mice had significantly fewer iNKT cells and normal numbers of T cells in the spleen compared with WT mice. In BALB/c VDR KO mice, the reduced frequencies of iNKT cells were not apparent in the liver or thymus. VDR KO and WT Th2 cells produced similar levels of IFN-γ and IL-5. On the BALB/c background, Th2 cells from VDR KO mice produced less IL-13, whereas on the C57BL/6 background, Th2 cells from VDR KO mice produced less IL-4. Conversely, VDR KO iNKT cells were defective for the production of multiple cytokines (BALB/c: IL-4, IL-5, and IL-13; C57BL/6: IL-4 and IL-17). Despite relatively normal Th2 responses, BALB/c and C57BL/6 VDR KO mice failed to develop AHR responses. The defect in iNKT cells as a result of the VDR KO was more important than the highly susceptible Th2 background of the BALB/c mice. Defective iNKT cell responses in the absence of the VDR result in the failure to generate AHR responses in the lung. The implication of these mechanistic findings for human asthma requires further investigation.     

ENA-A actimineral resource A extends lifespan associated with antioxidant mechanism in SMP30 knockout mice

ENA-actimineral resource A (ENA-A) is an alkaline mineral water and has a few biological activities such as antioxidant activity. The aim of this study was to examine the effects of ENA-A on lifespan in mice using senescence marker protein-30 knockout mice. The present study had groups of 18-week-old mice (n = 24), 26-week-old mice (n = 12), and 46-week-old mice (n = 20). Each differently aged mice group was divided into three subgroups: a control group, a 5 % ENA-A-treated group, and a 10 % ENA-A-treated group. Mice in the 18-week-old group were treated with vitamin C drinking water 1.5 g/L. However, the mice in the 26-week-old and 46-week-old groups were not treated with vitamin C. The experiments were done for 18 weeks. All vitamin C-treated mice were alive at week 18 (100 % survival rate). In the non-vitamin C group, the 10 % ENA-A-treated mice were alive at week 18. The control and 5 % ENA-A-treated mice died by week 15. As expected, vitamin C was not detected in the non-vitamin C-treated group. However, vitamin C levels were increased in an ENA-A dose-dependent manner in the vitamin C-treated group. In the TUNEL assay, a number of positive hepatocytes significantly decreased in an ENA-A dose-dependent manner. Periodic acid Schiff positive hepatocytes were significantly increased in an ENA-A dose-dependent manner. In addition, the expression level of CuZnSOD was increased by the ENA-A treatment. These data suggest that the intake of ENA-A has a critical role in the anti-aging mechanism and could be applied toward the lifespans of humans.     

Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects

Smad5 has been implicated as a downstream signal mediator for several bone morphogenetic proteins (BMPs). To understand the in vivo function of Smad5, we generated mice deficient in Smad5 using embryonic stem (ES) cell technology. Homozygous mutant embryos die between E9.5 and E11.5, and display variable phenotypes. Morphological defects are first detected at E8.0 in the developing amnion, gut and heart (the latter defect being similar to BMP-2 knockout mice). At later stages, mutant embryos fail to undergo proper turning, have craniofacial and neural tube abnormalities, and are edematous. In addition, several extraembryonic lesions are observed. After E9.0, the yolk sacs of the mutants contain red blood cells but lack a well-organized vasculature, which is reminiscent of BMP-4, TGF-beta1 and TGF-beta type II receptor knockout mice. In addition, the allantois of many Smad5 mutants is fused to the chorion, but is not well-elongated. A unique feature of the Smad5 mutant embryos is that ectopic vasculogenesis and hematopoiesis is observed in the amnion, likely due to mislocation of allantois tissue. Despite the expression of Smad5 from gastrulation onwards, and in contrast to knockouts of Smad2 and Smad4, Smad5 only becomes essential later in extraembryonic and embryonic development.