Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research

Background

Mice expressing fluorescent proteins in neurons are one of the most powerful tools in modern neuroscience research and are increasingly being used for in vivo studies of neurodegeneration. However, these mice are often used under the assumption that the fluorescent proteins present are biologically inert.

Methodology/Principal Findings

Here, we show that thy1-driven expression of yellow fluorescent protein (YFP) in neurons triggers multiple cell stress responses at both the mRNA and protein levels in vivo. The presence of YFP in neurons also subtly altered neuronal morphology and modified the time-course of dying-back neurodegeneration in experimental axonopathy, but not in Wallerian degeneration triggered by nerve injury.

Conclusions/Significance

We conclude that fluorescent protein expressed in thy1-YFP mice is not biologically inert, modifies molecular and cellular characteristics of neurons in vivo, and has diverse and unpredictable effects on neurodegeneration pathways.     

Messenger RNA oxidation occurs early in disease pathogenesis and promotes motor neuron degeneration in ALS

Background

Accumulating evidence indicates that RNA oxidation is involved in a wide variety of neurological diseases and may be associated with neuronal deterioration during the process of neurodegeneration. However, previous studies were done in postmortem tissues or cultured neurons. Here, we used transgenic mice to demonstrate the role of RNA oxidation in the process of neurodegeneration.

Methodology/Principal Findings

We demonstrated that messenger RNA (mRNA) oxidation is a common feature in amyotrophic lateral sclerosis (ALS) patients as well as in many different transgenic mice expressing familial ALS-linked mutant copper-zinc superoxide dismutase (SOD1). In mutant SOD1 mice, increased mRNA oxidation primarily occurs in the motor neurons and oligodendrocytes of the spinal cord at an early, pre-symptomatic stage. Identification of oxidized mRNA species revealed that some species are more vulnerable to oxidative damage, and importantly, many oxidized mRNA species have been implicated in the pathogenesis of ALS. Oxidative modification of mRNA causes reduced protein expression. Reduced mRNA oxidation by vitamin E restores protein expression and partially protects motor neurons.

Conclusion/Significance

These findings suggest that mRNA oxidation is an early event associated with motor neuron deterioration in ALS, and may be also a common early event preceding neuron degeneration in other neurological diseases.     

Adherent monomer-misfolded SOD1

Background

Multiple cellular functions are compromised in amyotrophic lateral sclerosis (ALS). In familial ALS (FALS) with Cu/Zn superoxide dismutase (SOD1) mutations, the mechanisms by which the mutation in SOD1 leads to such a wide range of abnormalities remains elusive.

Methodology/Principal Findings

To investigate underlying cellular conditions caused by the SOD1 mutation, we explored mutant SOD1-interacting proteins in the spinal cord of symptomatic transgenic mice expressing a mutant SOD1, SOD1^Leu126delTT with a FLAG sequence (DF mice). This gene product is structurally unable to form a functional homodimer. Tissues were obtained from both DF mice and disease-free mice expressing wild-type with FLAG SOD1 (WF mice). Both FLAG-tagged SOD1 and cross-linking proteins were enriched and subjected to a shotgun proteomic analysis. We identified 34 proteins (or protein subunits) in DF preparations, while in WF preparations, interactions were detected with only 4 proteins.

Conclusions/Significance

These results indicate that disease-causing mutant SOD1 likely leads to inadequate protein-protein interactions. This could be an early and crucial process in the pathogenesis of FALS.     

Novel Oxytocin Gene Expression in the Hindbrain Is Induced by Alcohol Exposure: Transgenic Zebrafish Enable Visualization of Sensitive Neurons

Background

Fetal Alcohol Spectrum Disorders (FASD) are a collection of disorders resulting from fetal ethanol exposure, which causes a wide range of physical, neurological and behavioral deficits including heightened susceptibility for alcoholism and addictive disorders. While a number of mechanisms have been proposed for how ethanol exposure disrupts brain development, with selective groups of neurons undergoing reduced proliferation, dysfunction and death, the induction of a new neurotransmitter phenotype by ethanol exposure has not yet been reported.

Principal Findings

The effects of embryonic and larval ethanol exposure on brain development were visually monitored using transgenic zebrafish expressing cell-specific green fluorescent protein (GFP) marker genes. Specific subsets of GFP-expressing neurons were highly sensitive to ethanol exposure, but only during defined developmental windows. In the med12 mutant, which affects the Mediator co-activator complex component Med12, exposure to lower concentrations of ethanol was sufficient to reduce GFP expression in transgenic embryos. In transgenic embryos and larva containing GFP driven by an oxytocin-like (oxtl) promoter, ethanol exposure dramatically up-regulated GFP expression in a small group of hindbrain neurons, while having no effect on expression in the neuroendocrine preoptic area.

Conclusions

Alcohol exposure during limited embryonic periods impedes the development of specific, identifiable groups of neurons, and the med12 mutation sensitizes these neurons to the deleterious effects of ethanol. In contrast, ethanol exposure induces oxtl expression in the hindbrain, a finding with profound implications for understanding alcoholism and other addictive disorders.     

NMDA Receptors on Non-Dopaminergic Neurons in the VTA Support Cocaine Sensitization

Background

The initiation of behavioral sensitization to cocaine and other psychomotor stimulants is thought to reflect N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic plasticity in the mesolimbic dopamine (DA) circuitry. The importance of drug induced NMDAR mediated adaptations in ventral tegmental area (VTA) DA neurons, and its association with drug seeking behaviors, has recently been evaluated in Cre-loxp mice lacking functional NMDARs in DA neurons expressing Cre recombinase under the control of the endogenous dopamine transporter gene (NR1^DATCre mice).

Methodology and Principal Findings

Using an additional NR1^DATCre mouse transgenic model, we demonstrate that while the selective inactivation of NMDARs in DA neurons eliminates the induction of molecular changes leading to synaptic strengthening, behavioral measures such as cocaine induced locomotor sensitization and conditioned place preference remain intact in NR1^DATCre mice. Since VTA DA neurons projecting to the prefrontal cortex and amygdala express little or no detectable levels of the dopamine transporter, it has been speculated that NMDA receptors in DA neurons projecting to these brain areas may have been spared in NR1^DATCre mice. Here we demonstrate that the NMDA receptor gene is ablated in the majority of VTA DA neurons, including those exhibiting undetectable DAT expression levels in our NR1^DATCre transgenic model, and that application of an NMDAR antagonist within the VTA of NR1^DATCre animals still blocks sensitization to cocaine.

Conclusions/Significance

These results eliminate the possibility of NMDAR mediated neuroplasticity in the different DA neuronal subpopulations in our NR1^DATCre mouse model and therefore suggest that NMDARs on non-DA neurons within the VTA must play a major role in cocaine-related addictive behavior.     

Recombinant Human Endostatin Endostar Suppresses Angiogenesis and Lymphangiogenesis of Malignant Pleural Effusion in Mice

Background

Malignant pleural effusion (MPE) is a common complication of lung cancer. One widely used treatment for MPE is Endostar, a recombined humanized endostatin based treatment. However, the mechanism of this treatment is still unclear. The aim of this study was to investigate the effects of Endostar in mice with MPE.

Methods and Materials

Lewis lung carcinoma (LLC) cell line expressing enhanced green fluorescent protein (EGFP) was injected into pleural cavity to establish MPE mice model. Mice were randomly divided into four groups. High dose of Endostar (30 mg/kg), low dose of Endostar (8 mg/kg), normal saline, or Bevacizumab (5 mg/kg) was respectively injected into pleural cavity three times with 3-day interval in each group. Transverse computed tomography (CT) was performed to observe pleural fluid formation 14 days after LLC cells injection. Mice were anesthetized and sacrificed 3 days after final administration. The volume of pleural effusion n was measured using 1 ml syringe. Micro blood vessel density (MVD), Lymphatic micro vessel density (LMVD), the expression level of vascular endothelial growth factor A (VEGF-A) and VEGF-C were observed by immunohistochemistry (IHC) staining.

Results

The volume of pleural effusion as well as the number of pleural tumor foci, MVD and the expression of VEGF-A were significantly reduced in high dose of Endostar treat group. More importantly, LMVD and the expression of VEGF-C were markedly lower in treat group than those in the other three control groups.

Conclusion

Our work demonstrated that Endostar played an efficient anti-cancer role in MPE through its suppressive effect on angiogenesis and lymphangiogenesis, which provided a certain theoretical basis for the effectiveness of Endostar on the MPE treatment.     

Neuroglobin Over Expressing Mice: Expression Pattern and Effect on Brain Ischemic Infarct Size

Background

Stroke is a major cause of death and severe disability, but effective treatments are limited. Neuroglobin, a neuronal heme-globin, has been advocated as a novel pharmacological target in combating stroke and neurodegenerative disorders based on cytoprotective properties. Using thoroughly validated antibodies and oligos, we give a detailed brain anatomical characterization of transgenic mice over expressing Neuroglobin. Moreover, using permanent middle artery occlusion the effect of elevated levels of Neuroglobin on ischemic damage was studied. Lastly, the impact of mouse strain genetic background on ischemic damage was investigated.

Principal Findings

A four to five fold increase in Neuroglobin mRNA and protein expression was seen in the brain of transgenic mice. A β-actin promoter was used to drive Neuroglobin over expression, but immunohistochemistry and in situ hybridization showed over expression to be confined to primarily the cortex, hippocampus, cerebellum, and only in neurons. The level and expression pattern of endogenous Neuroglobin was unaffected by insertion of the over expressing Ngb transgene. Neuroglobin over expression resulted in a significant reduction in infarct volume 24 hours after ischemia. Immunohistochemistry showed no selective sparing of Neuroglobin expressing cells in the ischemic core or penumbra. A significant difference in infarct volume was found between mice of the same strain, but from different colonies.

Significance

In contrast to some previous reports, Neuroglobin over expression is not global but confined to a few well-defined brain regions, and only in neurons. This study confirms previous reports showing a correlation between reduced infarct volume and elevated Neuroglobin levels, but underlines the need to study the likely contribution from compensatory mechanisms to the phenotype following a genetic perturbation. We also stress, that care should be taken when comparing results where different mouse strains and colonies have been used due to large genetic background contribution to the observed phenotype.     

Hilar GABAergic interneuron activity controls spatial learning and memory retrieval

Background

Although extensive research has demonstrated the importance of excitatory granule neurons in the dentate gyrus of the hippocampus in normal learning and memory and in the pathogenesis of amnesia in Alzheimer''s disease (AD), the role of hilar GABAergic inhibitory interneurons, which control the granule neuron activity, remains unclear.

Methodology and Principal Findings

We explored the function of hilar GABAergic interneurons in spatial learning and memory by inhibiting their activity through Cre-dependent viral expression of enhanced halorhodopsin (eNpHR3.0)—a light-driven chloride pump. Hilar GABAergic interneuron-specific expression of eNpHR3.0 was achieved by bilaterally injecting adeno-associated virus containing a double-floxed inverted open-reading frame encoding eNpHR3.0 into the hilus of the dentate gyrus of mice expressing Cre recombinase under the control of an enhancer specific for GABAergic interneurons. In vitro and in vivo illumination with a yellow laser elicited inhibition of hilar GABAergic interneurons and consequent activation of dentate granule neurons, without affecting pyramidal neurons in the CA3 and CA1 regions of the hippocampus. We found that optogenetic inhibition of hilar GABAergic interneuron activity impaired spatial learning and memory retrieval, without affecting memory retention, as determined in the Morris water maze test. Importantly, optogenetic inhibition of hilar GABAergic interneuron activity did not alter short-term working memory, motor coordination, or exploratory activity.

Conclusions and Significance

Our findings establish a critical role for hilar GABAergic interneuron activity in controlling spatial learning and memory retrieval and provide evidence for the potential contribution of GABAergic interneuron impairment to the pathogenesis of amnesia in AD.     

A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination

The success of Cre-mediated conditional gene targeting depends on the specificity of Cre recombinase expression in Cre-transgenic mouse lines. As a tool to evaluate the specificity of Cre expression, we developed a reporter transgenic mouse strain that expresses enhanced green fluorescent protein (EGFP) upon Cre-mediated recombination. We demonstrate that the progeny resulting from a cross between this reporter strain and a transgenic strain expressing Cre in zygotes show ubiquitous EGFP fluorescence. This reporter strain should be useful to monitor the Cre expression directed by various promoters in transgenic mice, including mice in which Cre is expressed transiently during embryogenesis under a developmentally regulated promoter.     

Using GFP video to track 3D movement and conditional gene expression in free-moving flies

Background

In vivo imaging and quantification of fluorescent reporter molecules is increasingly useful in biomedical research. For example, tracking animal movement in 3D with simultaneous quantification of fluorescent transgenic reporters allows for correlations between behavior, aging and gene expression. However implementation has been hindered in the past by the complexity of operating the systems.

Results

We report significant technical improvements and user-friendly software (called FluoreScore) that enables tracking of 3D movement and the dynamics of gene expression in adult Drosophila, using two cameras and recorded GFP videos. Expression of a transgenic construct encoding eGFP was induced in free-moving adult flies using the Gene-Switch system and RU486 drug feeding. The time course of induction of eGFP expression was readily quantified from internal tissues including central nervous tissue.

Conclusions

FluoreScore should facilitate a variety of future studies involving quantification of movement behaviors and fluorescent molecules in free-moving animals.     

Aging Is Not Associated with Proteasome Impairment in UPS Reporter Mice

Background

Covalent linkage of ubiquitin regulates the function and, ultimately, the degradation of many proteins by the ubiquitin-proteasome system (UPS). Given its essential role in protein regulation, even slight perturbations in UPS activity can substantially impair cellular function.

Methodology/Principal Findings

We have generated and characterized a novel transgenic mouse model which expresses a previously described reporter for UPS function. This UPS reporter contains a degron sequence attached to the C-terminus of green fluorescent protein, and is predominantly expressed in neurons throughout the brain of our transgenic model. We then demonstrated that this reporter system is sensitive to UPS inhibition in vivo.

Conclusions/Significance

Given the obstacles associated with evaluating proteasomal function in the brain, our mouse model uniquely provides the capability to monitor UPS function in real time in individual neurons of a complex organism. Our novel mouse model now provides a useful resource with which to evaluate the impact of aging, as well as various genetic and/or pharmacological modifiers of neurodegenerative disease(s).     

Calreticulin Induces Dilated Cardiomyopathy

Background

Calreticulin, a Ca²⁺-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart.

Methodology/Principal Findings

We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca²⁺ signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca²⁺-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca²⁺-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin.

Conclusions/Significance

We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca²⁺ handling genes, gap junction components and left ventricle remodeling.     

A Novel Cre Recombinase Imaging System for Tracking Lymphotropic Virus Infection In Vivo

Background

Detection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells.

Methodology/Principal findings

Murine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow.

Conclusions/Significance

The use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections.     

Possible Roles of mmu-miR-141 in the Endometrium of Mice in Early Pregnancy Following Embryo Implantation

Objective

Embryo implantation is directly affected by genes related to uterine receptivity. Studies have demonstrated the important roles of miRNAs in the regulation of gene expression. Our early miRNA chip analyses revealed that the mmu-miR-141 expression in endometrial tissue is lower after embryo implantation than before it. However, the possible roles of miR-141 in embryo implantation have not yet been elucidated. Here, mmu-miR-141 was designed to detect the expression and role of miR-141 in the endometria of mice in early pregnancy following embryo implantation.

Methods

Real-time PCR and in-situ hybridization were used to study mmu-miR-141 expression in mouse uterus. Cell proliferation was detected by tetrazolium dye (MTT) assay and flow cytometry. Real-time PCR and Western blot analysis were used to confirm the mRNA and protein levels of phosphatase and tensin homolog (PTEN) to determine whether it was the target gene of mmu-miR-141. Enhanced green fluorescent protein (EGFP) fluorescence reporter vector analysis was also performed. A functional study was performed by injecting mice uteri with mmu-miR-141 inhibitor or mimic vectors.

Results

mmu-miR-141 expression was lower on day 6 (D6) than day 4 (D4) and could be increased by progesterone. Reduced mmu-miR-141 could decrease the proliferation activity of stromal cells and promote apoptosis. Upregulation of mmu-miR-141 inhibited PTEN protein expression but downregulation of mmu-miR-141 increased it, while the mRNA level remained unchanged. EGFP fluorescence reporter vector analysis showed that miR-141 targets the 3′-untranslated region of the PTEN mRNA. In addition, when the physiological mmu-miR-141 level was altered on D2 by injecting with inhibitor or mimic, the embryo implantation sites were significantly decreased on D7.

Conclusions

This study demonstrated that mmu-miR-141 might influence cell proliferation and apoptosis in the endometrium by negatively regulating PTEN expression, and could also influence the number of embryo implantation sites. mmu-miR-141 plays an essential role in embryo implantation.     

Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

Background

Low efficiency of gene transfer and silence of transgene expression are the critical factors hampering the development of transgenic livestock. Recently, transfer of recombinant lentivirus has been demonstrated to be an efficient transgene delivery method in various animals. However, the lentiviral transgenesis and the methylation status of transgene in sheep have not been well addressed.

Methodology/Principle Findings

EGFP transgenic sheep were generated by injecting recombinant lentivirus into zygotes. Of the 13 lambs born, 8 carried the EGFP transgene, and its chromosomal integration was identified in all tested tissues. Western blotting showed that GFP was expressed in all transgenic founders and their various tissues. Analysis of CpG methylation status of CMV promoter by bisulfate sequencing unraveled remarkable variation of methylation levels in transgenic sheep. The average methylation levels ranged from 37.6% to 79.1% in the transgenic individuals and 34.7% to 83% in the tested tissues. Correlative analysis of methylation status with GFP expression revealed that the GFP expression level was inversely correlated with methylation density. The similar phenomenon was also observed in tested tissues. Transgene integration determined by Southern blotting presented multiple integrants ranging from 2 to 6 copies in the genome of transgenic sheep.

Conclusions/Significance

Injection of lentiviral transgene into zygotes could be a promising efficient gene delivery system to generate transgenic sheep and achieved widespread transgene expression. The promoter of integrants transferred by lentiviral vector was subjected to dramatic alteration of methylation status and the transgene expression level was inversely correlative with promoter methylation density. Our work illustrated for the first time that generation of transgenic sheep by injecting recombinant lentivirus into zygote could be an efficient tool to improve sheep performance by genetic modification.