Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon

We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.     

Severe hepatocellular disease in mice lacking one or both CaaX prenyltransferases

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) add 15- or 20-carbon lipids, respectively, to proteins that terminate with a CaaX motif. These posttranslational modifications of proteins with lipids promote protein interactions with membrane surfaces in cells, but the in vivo importance of the CaaX prenyltransferases and the protein lipidation reactions they catalyze remain incompletely defined. One study concluded that a deficiency of FTase was inconsequential in adult mice and led to little or no tissue pathology. To assess the physiologic importance of the CaaX prenyltransferases, we used conditional knockout alleles and an albumin-Cre transgene to produce mice lacking FTase, GGTase-I, or both enzymes in hepatocytes. The hepatocyte-specific FTase knockout mice survived but exhibited hepatocellular disease and elevated transaminases. Mice lacking GGTase-I not only had elevated transaminases but also had dilated bile cannaliculi, hyperbilirubinemia, hepatosplenomegaly, and reduced survival. Of note, GGTase-I-deficient hepatocytes had a rounded shape and markedly reduced numbers of actin stress fibers. Hepatocyte-specific FTase/GGTase-I double-knockout mice closely resembled mice lacking GGTase-I alone, but the disease was slightly more severe. Our studies refute the notion that FTase is dispensable and demonstrate that GGTase-I is crucial for the vitality of hepatocytes.     

Interferon-gamma production and host protective response against Mycobacterium tuberculosis in mice lacking both IL-12p40 and IL-18

Interferon (IFN)-gamma plays an essential role in host defense against infection with Mycobacterium tuberculosis, and its synthesis is critically regulated by interleukin (IL)-12, IL-18 and the recently identified IL-23. The present study was designed to determine the roles of these cytokines in IFN-gamma-mediated host defenses against M. tuberculosis. For this purpose, we compared host protective responses in IL-12p40 and IL-18 double-knockout (DKO) mice (which lacked both IL-12/IL-18 and also IL-23) and IFN-gamma gene-disrupted (GKO) mice. DKO mice were more resistant to the infection than GKO mice, as indicated by their extended survival and reduced live colony numbers in spleen, liver and lung. IFN-gamma was detected by ELISA in liver and lung homogenates, but not in spleen and serum, and in all organs by RT-PCR in DKO mice at comparable or reduced levels to those in wild-type mice. IFN-gamma production was reduced by depletion of CD4+ T cells, but not of natural killer (NK), NKT, gammadeltaT and dendritic cells. Neutralization of IFN-gamma or TNF-alpha by specific monoclonal antibodies (mAbs) significantly shortened the survival time of the infected DKO mice. Furthermore, anti-TNF-alpha mAb partially attenuated IFN-gamma synthesis in the liver of these mice. Finally, the expression level of inducible nitric oxide synthase (iNOS) mRNA in the spleen, liver and lung was considerable in DKO mice but only marginal or undetected in GKO mice. Our results indicate the presence of IL-12-, IL-18- and IL-23-independent host protective responses against mycobacterial infection mediated by IFN-gamma, which was secreted from helper T cells.     

Renal function of gene-targeted mice lacking both SGK1 and SGK3

Serum- and glucocorticoid-inducible kinase (SGK) 1 and SGK3 share the ability to upregulate several ion channels, including the epithelial Na(+) channel. Whereas SGK1 is under genomic control of mineralocorticoids and glucocorticoids, SGK3 is constitutively expressed. The SKG1-knockout (sgk1(-/-)) mouse is seemingly normal when it is fed a standard diet, but its ability to retain NaCl is impaired when it is fed a salt-deficient diet. In the SGK3-knockout (sgk3(-/-)) mouse fed standard and salt-deficient diets, hair growth is strikingly delayed but NaCl excretion is normal. Thus the possibility was considered that SGK1 and SGK3 could mutually replace each other, thus preventing severe NaCl loss in sgk1(-/-) and sgk3(-/-) mice. We crossed SGK1- and SGK3-knockout mice and compared renal electrolyte excretion of the double mutants (sgk1(-/-)/sgk3(-/-)) with that of their wild-type littermates (sgk1(+/+)/sgk3(+/+)). Similar to sgk3(-/-) mice, the sgk1(-/-)/sgk3(-/-) mice display delayed hair growth. Blood pressure was slightly, but significantly (P < 0.03), lower in sgk1(-/-)/sgk3(-/-) (102 +/- 4 mmHg) than in sgk1(+/+)/sgk3(+/+) (114 +/- 3 mmHg) mice, a difference that was maintained in mice fed low- and high-salt diets. Plasma aldosterone concentrations were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) than in sgk1(+/+)sgk3(+/+) mice fed control (511 +/- 143 vs. 143 +/- 32 pg/ml) and low-salt (1,325 +/- 199 vs. 362 +/- 145 pg/ml) diets. During salt depletion, absolute and fractional excretions of Na(+) were significantly (P < 0.01) higher in sgk1(-/-)/sgk3(-/-) (1.2 +/- 0.2 micromol/24 h g body wt, 0.12 +/- 0.03%) than in sgk1(+/+)/sgk3(+/+) (0.4 +/- 0.1 micromol/24 h g body wt, 0.04 +/- 0.01%) mice. The sgk1(-/-)/sgk3(-/-) mice share the delayed hair growth with sgk3(-/-) mice and the modestly impaired renal salt retention with sgk1(-/-) mice. Additional lack of the isoform kinase does not substantially compound the phenotype for either property.     

Central adaptation models of the vestibulo-ocular and optokinetic systems

A theoretical analysis of two models of the vestibulo-ocular and optokinetic systems was performed. Each model contains a filter element in the vestibular periphery to account for peripheral adaptation, and a filter element in the central vestibulooptokinetic circuit to account for central adaptation. Both models account for1 adaptation, i.e. a response decay to a constant angular acceleration input, in both peripheral vestibular afferent and vestibulo-ocular reflex (VOR) responses and2 the reversal phases of optokinetic after-nystagmus (OKAN) and the VOR and3 oscillatory behavior such as periodic alternating nystagmus. The two models differ regarding the order of their VOR transfer function. Also, they predict different OKAN patterns following a prolonged optokinetic stimulus. These models have behavioral implications and suggest future experiments.     

Wip1 phosphatase modulates both long-term potentiation and long-term depression through the dephosphorylation of CaMKII

Synaptic plasticity is an important mechanism that underlies learning and cognition. Protein phosphorylation by kinases and dephosphorylation by phosphatases play critical roles in the activity-dependent alteration of synaptic plasticity. In this study, we report that Wip1, a protein phosphatase, is essential for long-term potentiation (LTP) and long-term depression (LTD) processes. Wip1-deletion suppresses LTP and enhances LTD in the hippocampus CA1 area. Wip1 deficiency-induced aberrant elevation of CaMKII T286/287 and T305 phosphorylation underlies these dysfunctions. Moreover, we showed that Wip1 modulates CaMKII dephosphorylation. Wip1^?/? mice exhibit abnormal GluR1 membrane expression, which could be reversed by the application of a CaMKII inhibitor, indicating that Wip1/CaMKII signaling is crucial for synaptic plasticity. Together, our results demonstrate that Wip1 phosphatase plays a vital role in regulating hippocampal synaptic plasticity by modulating the phosphorylation of CaMKII.     

Augmented atherogenesis in LDL receptor deficient mice lacking both macrophage ABCA1 and ApoE

Aim

ABCA1 protects against atherosclerosis by facilitating cholesterol efflux from macrophage foam cells in the arterial wall to extracellular apolipoprotein (apo) A-I. In contrast to apoA-I, apoE is secreted by macrophages and can, like apoA-I, induce ABCA1-mediated cholesterol efflux. Yet, the combined effect of macrophage ABCA1 and apoE on lesion development is unexplored.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/apoE double KO (dKO) mice, their respective single KO''s, and wild-type (WT) controls and were challenged with a high-fat/high-cholesterol diet for 9 weeks. In vitro cholesterol efflux experiments showed no differences between ABCA1 KO and dKO macrophages. The serum non-HDL/HDL ratio in dKO transplanted mice was 1.7-fold and 2.4-fold (p<0.01) increased compared to WT and ABCA1 KO transplanted mice, respectively. The atherosclerotic lesion area in dKO transplanted animals (650±94×10³ µm²), however, was 1.9-fold (p<0.01) and 1.6-fold (p<0.01) increased compared to single knockouts (ABCA1 KO: 341±20×10³ µm²; apoE KO: 402±78×10³ µm², respectively) and 3.1-fold increased (p<0.001) compared to WT (211±20×10³ µm²). When normalized for serum cholesterol exposure, macrophage ABCA1 and apoE independently protected against atherosclerotic lesion development (p<0.001). Moreover, hepatic expression levels of TNFα and IL-6 were highly induced in dKO transplanted animals (3.0-fold; p<0.05, and 4.3-fold; p<0.001, respectively). In agreement, serum IL-6 levels were also enhanced in ABCA1 KO transplanted mice (p<0.05) and even further enhanced in dKO transplanted animals (3.1-fold as compared to ABCA1 KO transplanted animals; p<0.05).

Conclusions

Combined deletion of macrophage ABCA1 and apoE results in a defect in cholesterol efflux and, compared to ABCA1 KO transplanted mice, elevated serum total cholesterol levels. Importantly, these mice also suffer from enhanced systemic and hepatic inflammation, together resulting in the observed augmented atherosclerotic lesion development.     

Mathematical simulation of induction of long-term depression in cerebellar Purkinje cells

Mechanisms of associative and homosynaptic long-term depression (LTD) in cerebellar Purkinje cells are discussed. The possibility of LTD induction related to a decrease in efficacy of AMPA receptors through either their dephosphorylation or phosphorylation is investigated by mathematical simulation.     

Selective deficits in the circadian light response in mice lacking PACAP

Previous studies indicate that light information reaches the suprachiasmatic nucleus through a subpopulation of retinal ganglion cells that contain both glutamate and pituitary adenylyl cyclase-activating peptide (PACAP). Although the role of glutamate in this pathway has been well studied, the involvement of PACAP and its receptors is only beginning to be understood. To investigate the functions of PACAP in vivo, we developed a mouse model in which the gene coding for PACAP was disrupted by targeted homologous recombination. RIA was used to confirm a lack of detectable PACAP protein in these mice. PACAP-deficient mice exhibited significant impairment in the magnitude of the response to brief light exposures with both light-induced phase delays and advances of the circadian system impacted. This mutation equally impacted phase shifts induced by bright and dim light exposure. Despite these effects on phase shifting, the loss of PACAP had only limited effects on the generation of circadian oscillations, as measured by rhythms in wheel-running activity. Unlike melanopsin-deficient mice, the mice lacking PACAP exhibited no loss of function in the direct light-induced inhibition of locomotor activity, i.e., masking. Finally, the PACAP-deficient mice exhibited normal phase shifts in response to exposure to discrete dark treatments. The results reported here show that the loss of PACAP produced selective deficits in the light response of the circadian system.     

Increased response to morphine in mice lacking protein kinase C epsilon

The protein kinase C (PKC) family of serine-threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCepsilon have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCepsilon null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCepsilon null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of mu-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCepsilon as a key regulator of opiate sensitivity in mice.     

Immunoproteasome assembly and antigen presentation in mice lacking both PA28alpha and PA28beta.

Two members of the proteasome activator, PA28alpha and PA28beta, form a heteropolymer that binds to both ends of the 20S proteasome. Evidence in vitro indicates that this interferon-gamma (IFN-gamma)-inducible heteropolymer is involved in the processing of intracellular antigens, but its functions in vivo remain elusive. To investigate the role of PA28alpha/beta in vivo, we generated mice deficient in both PA28alpha and PA28beta genes. The ATP-dependent proteolytic activities were decreased in PA28alpha(-/-)/beta(-/-) cells, suggesting that 'hybrid proteasomes' are involved in protein degradation. Treatment of PA28alpha(-/-)/beta(-/-) cells with IFN-gamma resulted in sufficient induction of the 'immunoproteasome'. Moreover, splenocytes from PA28alpha(-/-)/beta(-/-) mice displayed no apparent defects in processing of ovalbumin. These results are in marked contrast to the previous finding that immunoproteasome assembly and immune responses were impaired in PA28beta(-/-) mice. PA28alpha(-/-)/beta(-/-) mice also showed apparently normal immune responses against infection with influenza A virus. However, they almost completely lost the ability to process a melanoma antigen TRP2-derived peptide. Hence, PA28alpha/beta is not a prerequisite for antigen presentation in general, but plays an essential role for the processing of certain antigens.     

Lethal hypoglycemic ketosis and glyceroluria in mice lacking both the mitochondrial and the cytosolic glycerol phosphate dehydrogenases

The activities of either the mitochondrial or cytosolic glycerol phosphate dehydrogenase (mGPD, cGPD) plus that of glycerol kinase are required for the use of glycerol in aerobic metabolism and gluconeogenesis. A knockout mouse lacking mGPD has reduced body weight and fertility but shows remarkably normal liver and muscle metabolite levels. The BALB/cHeA mouse strain, which lacks cGPD, breeds well and is phenotypically normal, although it demonstrates metabolite abnormalities in certain tissues. Crosses were made between these two strains, and mice were generated that lacked both dehydrogenases. These mice, although active and nursing well for several days, failed to grow, and usually died within the first week. Liver glycerol phosphate levels were elevated 30-fold, whereas liver ATP, ADP, and AMP levels were reduced by 30-40%. Plasma glycerol was elevated 30- to 50-fold to 30-50 mm, and urine glycerol exceeded 0.45 m (4% w/v). GPD-deficient mice were hypoglycemic, had a 50% increase in plasma free fatty acids, and developed ketonuria within the first day of life. Uncoupling protein-1 mRNA in brown adipose tissue was reduced 60%. These mice share some features of both glycerol kinase deficiency and hereditary fructose intolerance, suggesting the phenotype may be due to the combined effects of the loss of a gluconeogenic substrate, the osmotic effects of glycerol, and the metabolic effects of the accumulation of a phosphorylated metabolite.     

Cardiovascular and metabolic alterations in mice lacking both beta1- and beta2-adrenergic receptors.

The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.     

A unique PDZ ligand in PKCalpha confers induction of cerebellar long-term synaptic depression

Induction of cerebellar long-term depression (LTD) requires a postsynaptic cascade involving activation of mGluR1 and protein kinase C (PKC). Our understanding of this process has been limited by the fact that PKC is a large family of molecules, many isoforms of which are expressed in the relevant postsynaptic compartment, the cerebellar Purkinje cell. Here, we report that LTD is absent in Purkinje cells in which the alpha isoform of PKC has been reduced by targeted RNA interference or in cells derived from PKCalpha null mice. In both of these cases, LTD could be rescued by expression of PKCalpha but not other PKC isoforms. The special role of PKCalpha in cerebellar LTD is likely to derive from its unique PDZ ligand (QSAV). When this motif is mutated, PKCalpha no longer supports LTD. Conversely, when this PDZ ligand is inserted in a nonpermissive isoform, PKCgamma, it confers the capacity for LTD induction.     

Platelet-activating factor receptor modulates respiratory adaptation to long-term intermittent hypoxia in mice

During hypoxia, release of platelet-activating factor (PAF) and activation of its cognate receptor (PAFR) regulate neural transmission and are required for full expression of peak hypoxic ventilatory response (pHVR) but not hypercapnic ventilatory response. However, it is unclear whether PAFR underlie components of long-term ventilatory adaptations to hypoxia. To examine this issue, adult male PAFR(+/+) and PAFR(-/-) mice were exposed to intermittent hypoxia (IH) consisting of 90 s 21% O(2) and 90 s 10% O(2) for 30 days, and normoxic and hypoxic ventilatory patterns were assessed using whole body plethysmography. Starting at day 14 of IH, normoxic ventilation in PAFR(-/-) was reduced significantly compared with PAFR(+/+) mice (P < 0.001), the latter exhibiting a prominent long-term ventilatory facilitation (LTVF). However, IH-exposed PAFR(-/-) mice had markedly enhanced pHVR and hypoxic ventilatory decline that became similar to those of IH-exposed PAFR(+/+) mice. Thus we postulate that PAFR expression and/or function underlies critical components of IH-induced LTVF but does not play a role in the potentiation of the hypoxic ventilatory response after IH exposures.     

B-cell-mediated depression of the granulomatous response to BCG in mice

The depression of the granulomatous response to Mycobacterium bovis strain BCG in mice infected intravenously with 2 x 10(7) CFU of the microorganism turned out to be mediated by various types of cells arising at different times after infection. Anti-PPD B lymphocytes were found to play a major role at Day 1 after infection and to be no longer effective 4 days later. At this time the depression was mediated by anti-idiotype B lymphocytes, whereas T lymphocytes proved to be involved in later phases of the infectious process. These results show that B lymphocytes may be of critical importance in the regulation of cell-mediated immune reactions to this facultative intracellular parasite.     

Measuring the optokinetic response of zebrafish larvae

Our laboratory screens for visual mutants by examining larval eye movements in response to rotating illuminated stripes. This behavior, which is termed an optokinetic response (OKR), is a reflex that appears in zebrafish at the same time as the development of the visual system. The OKR can be accurately measured by 4 d post-fertilization, which is the age when larvae begin foraging for food. The OKR requires approximately 1 min per larva analyzed. After identifying fish with defective eye movements, we conduct secondary screens (such as histological analysis and electroretinography) to identify the subset of fish with disruptions in the function of the outer retina. This paper describes our protocol for the OKR. Our setup is simple to construct and the materials needed are inexpensive. This makes our system especially useful for new undergraduate and graduate students, as well as introductory science lecturers.     

诱导成年大鼠海马CA1区长时程压抑的强直刺激型式

标准低频率连续刺激(1～2 Hz,15 min)能够诱导幼年大鼠(＜4周)海马CA1区同突触长时程压抑(long-term depression,LTD),而只有较高频率且持续时间较长的连续刺激才能诱导出成年动物该部位稳定的LTD.本研究采用成年大鼠海马脑片标本,电刺激Schaffer侧枝传入纤维,在CA1区锥体细胞层记录群体锋电位,选用两种新的刺激参数以观测不同刺激型式在诱导成年大鼠LTD中的作用.诱导LTD的刺激参数为(1)2 Hz,5串,串长60 s,串间隔60 s;(2)5 Hz,5串,串长24 s,串间隔96 s;(3)对照组参数2 Hz,300 s.结果显示,对照参数未能诱导出LTD;而两种频率不同但脉冲总数与刺激总时程相同的多串刺激,即参数(1)与参数(2),均在成年大鼠海马CA1区诱导产生了LTD.两种参数所诱导的LTD特征具有参数特异性,该特征主要表现为LTD诱导潜伏期和LTD的幅度参数(1)、(2)诱导的LTD的潜伏期分别为15～25 min和30～40 min;强直刺激后80 min时LTD的幅度分别为(57.5±2.8)%和(67.7±3.4)%.以上结果表明特定型式的低频率刺激能够诱导成年大鼠海马CA1区的LTD,提示LTD的诱导与刺激的组合型式相关,并且2 Hz较5 Hz的多串刺激在诱导LTD中更为有效.