MRI follow-up of TNF-dependent differential progression of atherosclerotic wall-thickening in mouse aortic arch from early to advanced stages

OBJECTIVES: An optimized, longitudinal in vivo magnetic resonance vessel wall-imaging protocol was evaluated regarding its capability of detecting differences in the time-dependent atherosclerotic lesion progression in the aortic arch between ApoE(-/-) and double-deficient ApoE(-/-)/TNF(-/-) mice at comparatively early plaque development stages. MATERIALS AND METHODS: Seven ApoE(-/-) and seven ApoE(-/-)/TNF(-/-) female mice underwent MRI at 11.75 teslas at four stages up to 26 weeks of age. A double-gated spin-echo MRI sequence was used with careful perpendicular slice positioning to visualize the vessel wall of the ascending aortic arch. RESULTS: Wall-thickness progression measured with MRI was significant at 11 weeks of age in ApoE(-/-) mice, but only at 26 weeks in ApoE(-/-)/TNF(-/-) mice. A significant correlation was found between MRI wall-thickness and lesion area determined on histology. CONCLUSION: MRI was shown to be sensitive enough to reveal subtle genetically-induced differences in lesion progression at ages earlier than 25 weeks.

The double-stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation

PURPOSE: Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF-2alpha). There have been no reports to date about the role of PKR in radiation sensitivity. EXPERIMENTAL DESIGN: A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-kappaB (NF-kappaB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence. RESULTS: Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR(-/-) MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-kappaB and Akt because both NF-kappaB and Akt are activated after ionizing radiation in PKR+/+ but not PKR-/- cells. CONCLUSIONS: We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-kappaB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB2 receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB2 agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions.

Neuroretina specification in mouse embryos requires Six3-mediated suppression of Wnt8b in the anterior neural plate.

Retinal degeneration causes vision impairment and blindness in humans. If one day we are to harness the potential of stem cell-based cell replacement therapies to treat these conditions, it is imperative that we better understand normal retina development. Currently, the genes and mechanisms that regulate the specification of the neuroretina during vertebrate eye development remain unknown. Here, we identify sine oculis-related homeobox 3 (Six3) as a crucial player in this process in mice. In Six3 conditional-mutant mouse embryos, specification of the neuroretina was abrogated, but that of the retinal pigmented epithelium was normal. Conditional deletion of Six3 did not affect the initial development of the optic vesicle but did arrest subsequent neuroretina specification. Ectopic rostral expansion of Wnt8b expression was the major response to Six3 deletion and the leading cause for the specific lack of neuroretina, as ectopic Wnt8b expression in transgenic embryos was sufficient to suppress neuroretina specification. Using chromatin immunoprecipitation assays, we identified Six3-responsive elements in the Wnt8b locus and demonstrated that Six3 directly repressed Wnt8b expression in vivo. Our findings provide a molecular framework to the program leading to neuroretina differentiation and may be relevant for the development of novel strategies aimed at characterizing and eventually treating different abnormalities in eye formation.

Attenuation of fibrosis in vitro and in vivo with SPARC siRNA.

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.

Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1.

Familial hemiplegic migraine type 1 (FHM1) is an autosomal dominant subtype of migraine with aura that is associated with hemiparesis. As with other types of migraine, it affects women more frequently than men. FHM1 is caused by mutations in the CACNA1A gene, which encodes the alpha1A subunit of Cav2.1 channels; the R192Q mutation in CACNA1A causes a mild form of FHM1, whereas the S218L mutation causes a severe, often lethal phenotype. Spreading depression (SD), a slowly propagating neuronal and glial cell depolarization that leads to depression of neuronal activity, is the most likely cause of migraine aura. Here, we have shown that transgenic mice expressing R192Q or S218L FHM1 mutations have increased SD frequency and propagation speed; enhanced corticostriatal propagation; and, similar to the human FHM1 phenotype, more severe and prolonged post-SD neurological deficits. The susceptibility to SD and neurological deficits is affected by allele dosage and is higher in S218L than R192Q mutants. Further, female S218L and R192Q mutant mice were more susceptible to SD and neurological deficits than males. This sex difference was abrogated by ovariectomy and senescence and was partially restored by estrogen replacement, implicating ovarian hormones in the observed sex differences in humans with FHM1. These findings demonstrate that genetic and hormonal factors modulate susceptibility to SD and neurological deficits in FHM1 mutant mice, providing a potential mechanism for the phenotypic diversity of human migraine and aura.

Synaptic vesicle dynamics in mouse rod bipolar cells.

To better understand synaptic signaling at the mammalian rod bipolar cell terminal and pave the way for applying genetic approaches to the study of visual information processing in the mammalian retina, synaptic vesicle dynamics and intraterminal calcium were monitored in terminals of acutely isolated mouse rod bipolar cells and the number of ribbon-style active zones quantified. We identified a releasable pool, corresponding to a maximum of 7 s. The presence of a smaller, rapidly releasing pool and a small, fast component of refilling was also suggested. Following calcium channel closure, membrane surface area was restored to baseline with a time constant that ranged from 2 to 21 s depending on the magnitude of the preceding Ca2+ transient. In addition, a brief, calcium-dependent delay often preceded the start of onset of membrane recovery. Thus, several aspects of synaptic vesicle dynamics appear to be conserved between rod-dominant bipolar cells of fish and mammalian rod bipolar cells. A major difference is that the number of vesicles available for release is significantly smaller in the mouse rod bipolar cell, both as a function of the total number per neuron and on a per active zone basis.

Molecular events associated with trisomy of chromosome 7 in mouse skin chemical carcinogenesis

The primary objective of this study has been to investigate the effects at the molecular level of trisomy of mouse chromosome 7 in chemically induced skin tumors. It was previously proposed that the initiation event in the mouse skin carcinogenesis model is a heterozygous mutation of the Ha-ras-1 gene, mapped to chromosome 7. Previous studies in this laboratory identified trisomy 7 as one of the primary nonrandom cytogenetic abnormalities found in the majority of severely dysplastic papillomas and squamous cell carcinomas induced in SENCAR mice by an initiation-promotion protocol. Therefore, the first hypothesis tested was that trisomy 7 occurs by specific duplication of the chromosome carrying a mutated Ha-ras-1 allele. Results of a quantitative analysis of normal/mutated allelic ratios of the Ha-ras-1 gene confirmed this hypothesis, showing that most of the tumors exhibited overrepresentation of the mutated allele in the form of 1/2, 0/3, and 0/2 (normal/mutated) ratios. In addition, histopathological analysis of the tumors showed an apparent association between the degree of malignancy and the dosage of the mutated Ha-ras-1 allele. To determine the mechanism for loss of the normal Ha-ras-1 allele, found in 30% of the tumors, a comparison of constitutional and tumor genotypes was performed at different informative loci of chromosome 7. By combining Southern blot and polymerase chain reaction fragment length polymorphism analyses of DNAs extracted from squamous cell carcinomas, complete loss of heterozygosity was detected in 15 of 20 tumors at the Hbb locus, and in 5 of 5 tumors at the int-2 locus, both distal to Ha-ras-1. In addition, polymerase chain reaction analysis of DNA extracted from papillomas indicated that loss of heterozygosity occurs in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion, suggesting that this event may be associated to the acquisition of the malignant phenotype. Allelic dosage analysis of tumors that had become homozygous at Hbb but retained heterozygosis at Ha-ras-1, indicated that loss of heterozygosity on mouse chromosome 7 occurs by a mitotic recombination mechanism. Overall, these findings suggest the presence of a putative tumor suppressor locus on the 7F1-ter region of mouse chromosome 7. Thus, loss of function by homozygosis at this putative suppressor locus may complement activation of the Ha-ras-1 gene during tumor progression, and might be associated with the malignant conversion stage of mouse skin carcinogenesis. ^

Use of a hypomorphic allele of myogenin to analyze Myogenin-dependent processes in mouse development

Myogenin is a muscle-specific transcription factor essential for skeletal muscle differentiation. A severe reduction in the number of fused myotubes is seen in myogenin-null mice, and the expression of genes characteristic of differentiated skeletal muscle is reduced. Additionally, sternebrae defects are seen in myogenin-null mice, a secondary defect in the sternal cartilage precursors. Very little is known about the quantitative requirement for myogenin in muscle differentiation and thoracic skeletal development in vivo. In this thesis I describe experiments utilizing a mouse line harboring a hypomorphic allele of myogenin, generated by gene targeting techniques in embryonic stem cells. The nature of the hypomorphism was due to lowered levels of myogenin from this allele. In embryos homozygous for the hypomorphic allele, normal sternum formation and extensive muscle differentiation was observed. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable after birth. These results suggest skeletal muscle differentiation is highly sensitive to the absolute amounts of myogenin, and reveal distinct threshold requirements for myogenin in skeletal muscle differentiation, sternum formation, and viability in vivo. The hypomorphic allele was utilized as a genetically sensitized background to identify other components of myogenin-mediated processes. Using a candidate gene approach I crossed null mutations in MEF2C and MRF4 into the hypomorphic background and examined whether these mutations affected muscle differentiation and skeleton formation in the myogenin hypomorph. Although MEF2C mutation did not affect any phenotypes seen in the hypomorphic background, MRF4 was observed to be an essential component of myogenin-mediated processes of thoracic skeletal development. Additionally, the hypomorphic allele was very sensitive to genetic effects, suggesting the existence of mappable genetic modifiers of the hypomorphic allele of myogenin. ^

Metabolomics reveals trichloroacetate as a major contributor to trichloroethylene-induced metabolic alterations in mouse urine and serum

Trichloroethylene (TCE)-induced liver toxicity and carcinogenesis is believed to be mediated in part by activation of the peroxisome proliferator-activated receptor α (PPARα). However, the contribution of the two TCE metabolites, dichloroacetate (DCA) and trichloroacetate (TCA) to the toxicity of TCE, remains unclear. The aim of the present study was to determine the metabolite profiles in serum and urine upon exposure of mice to TCE, to aid in determining the metabolic response to TCE exposure and the contribution of DCA and TCA to TCE toxicity. C57BL/6 mice were administered TCE, TCA, or DCA, and urine and serum subjected to ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-based global metabolomics analysis. The ions were identified through searching metabolomics databases and by comparison with authentic standards, and quantitated using multiple reactions monitoring. Quantitative polymerase chain reaction of mRNA, biochemical analysis, and liver histology were also performed. TCE exposure resulted in a decrease in urine of metabolites involved in fatty acid metabolism, resulting from altered expression of PPARα target genes. TCE treatment also induced altered phospholipid homeostasis in serum, as revealed by increased serum lysophosphatidylcholine 18:0 and 18:1, and phosphatidylcholine metabolites. TCA administration revealed similar metabolite profiles in urine and serum upon TCE exposure, which correlated with a more robust induction of PPARα target gene expression associated with TCA than DCA treatment. These data show the metabolic response to TCE exposure and demonstrate that TCA is the major contributor to TCE-induced metabolite alterations observed in urine and serum.

Latrepirdine stimulates autophagy and reduces accumulation of α-synuclein in cells and in mouse brain

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aβ42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

Oxidative Stress and Ca2+ Release Events in Mouse Cardiomyocytes

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca2+ signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca2+ release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca2+ through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca2+ signaling in situ, by analyzing Ca2+ sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca2+ spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca2+ release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca2+ depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca2+ release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca2+ release events.

Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers

Antisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders1, 2. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin3, 4 and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure5 and neurocognitive impairment6. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.