A Comprehensive Review of Genetically Engineered Mouse Models for Prader-Willi Syndrome Research

Prader-Willi syndrome (PWS) is a neurogenetic multifactorial disorder caused by the deletion or inactivation of paternally imprinted genes on human chromosome 15q11-q13. The affected homologous locus is on mouse chromosome 7C. The positional conservation and organization of genes including the imprinting pattern between mice and men implies similar physiological functions of this locus. Therefore, considerable efforts to recreate the pathogenesis of PWS have been accomplished in mouse models. We provide a summary of different mouse models that were generated for the analysis of PWS and discuss their impact on our current understanding of corresponding genes, their putative functions and the pathogenesis of PWS. Murine models of PWS unveiled the contribution of each affected gene to this multi-facetted disease, and also enabled the establishment of the minimal critical genomic region (PWScr) responsible for core symptoms, highlighting the importance of non-protein coding genes in the PWS locus. Although the underlying disease-causing mechanisms of PWS remain widely unresolved and existing mouse models do not fully capture the entire spectrum of the human PWS disorder, continuous improvements of genetically engineered mouse models have proven to be very powerful and valuable tools in PWS research.     

The House Mouse (Mus musculus L.) Exerts Strong Differential Grain Consumption Preferences among Hard Red and White Spring Wheat (Triticum aestivum L.) Varieties in a Single‐Elimination Tournament Design

Wheat (Triticum aestivum L.) plays a central role in the health and nutrition of humans. Yet, little is known about possible flavor differences among different varieties. We have developed a model system using the house mouse (Mus musculus L.) to determine feeding preferences as a prelude to extending results to human sensory analysis. Here, we examine the application of a single‐elimination tournament design to the analysis of consumption preferences of a set of hard red and hard white spring wheat varieties. A single‐elimination tournament design in this case pairs 2 wheat varieties and only 1 of the 2 is advanced to further tests. Preferred varieties were advanced until an overall “winner” was identified; conversely, less desirable varieties were advanced such that an overall “loser” was identified. Hollis and IDO702 were the winner and loser, respectively, for the hard red varieties, and Clear White 515 and WA8123 were the winner and loser, respectively, for the hard white varieties. When using the more powerful protocol of 14 mice and a 4?d trial, differences in mean daily consumption preferences of 2 varieties were separated at P‐values as small as 2 × 10^?8. The single‐elimination tournament design is an efficient means of identifying the most and least desirable varieties among a larger set of samples. One application for identifying the 2 extremes in preference within a group of varieties would be to use them as parents of a population to identify quantitative trait loci for preference.     

枸杞多糖对顺铂诱导小鼠睾丸支持细胞凋亡的影响

目的：研究枸杞多糖（lycium barbarum polysaccharides, LBP）对顺铂（cis-dichlorodiamineplatinum, CDDP）诱导小鼠睾丸支持细胞（TM4）凋亡的影响并探讨其可能机制。方法：体外培养TM4细胞,四甲基偶氮噻唑蓝（MTT）法检测LBP对CDDP诱导TM4细胞生存率的影响,Western blot检测LBP对CDDP诱导TM4细胞凋亡相关基因Bcl-2、Bax和Caspase-3蛋白表达的影响,流式细胞仪检测细胞凋亡率的变化。结果：与Control组相比,CDDP组TM4细胞凋亡显著增加,细胞内抑凋亡基因Bcl-2表达量显著降低,促凋亡基因Bax表达量及Caspase-3表达量显著升高;与CDDP组相比,CDDP+LBP组TM4细胞凋亡显著减少,细胞内抑凋亡基因Bcl-2表达量显著升高,促凋亡基因Bax表达量及Caspase-3表达量显著降低。 结论：LBP作用于CDDP诱导的TM4细胞,能够通过增强细胞内Bcl-2的表达,抑制Bax及Caspase-3的表达来阻抑因CDDP诱导的TM4细胞凋亡,进而减轻CDDP对TM4细胞造成的损伤。     

High,in Contrast to Low Levels of Acute Stress Induce Depressive-like Behavior by Involving Astrocytic,in Addition to Microglial P2X7 Receptors in the Rodent Hippocampus

Extracellular adenosine 5′-triphosphate (ATP) in the brain is suggested to be an etiological factor of major depressive disorder (MDD). It has been assumed that stress-released ATP stimulates P2X7 receptors (Rs) at the microglia, thereby causing neuroinflammation; however, other central nervous system (CNS) cell types such as astrocytes also possess P2X7Rs. In order to elucidate the possible involvement of the MDD-relevant hippocampal astrocytes in the development of a depressive-like state, we used various behavioral tests (tail suspension test [TST], forced swim test [FST], restraint stress, inescapable foot shock, unpredictable chronic mild stress [UCMS]), as well as fluorescence immunohistochemistry, and patch-clamp electrophysiology in wild-type (WT) and genetically manipulated rodents. The TST and FST resulted in learned helplessness manifested as a prolongation of the immobility time, while inescapable foot shock caused lower sucrose consumption as a sign of anhedonia. We confirmed the participation of P2X7Rs in the development of the depressive-like behaviors in all forms of acute (TST, FST, foot shock) and chronic stress (UCMS) in the rodent models used. Further, pharmacological agonists and antagonists acted in a different manner in rats and mice due to their diverse potencies at the respective receptor orthologs. In hippocampal slices of mice and rats, only foot shock increased the current responses to locally applied dibenzoyl-ATP (Bz-ATP) in CA1 astrocytes; in contrast, TST and restraint depressed these responses. Following stressful stimuli, immunohistochemistry demonstrated an increased co-localization of P2X7Rs with a microglial marker, but no change in co-localization with an astroglial marker. Pharmacological damage to the microglia and astroglia has proven the significance of the microglia for mediating all types of depression-like behavioral reactions, while the astroglia participated only in reactions induced by strong stressors, such as foot shock. Because, in addition to acute stressors, their chronic counterparts induce a depressive-like state in rodents via P2X7R activation, we suggest that our data may have relevance for the etiology of MDD in humans.     

Interaction of Cucurbit[7]uril with Oxime K027, Atropine,and Paraoxon: Risky or Advantageous Delivery System?

Antidotes against organophosphates often possess physicochemical properties that mitigate their passage across the blood–brain barrier. Cucurbit[7]urils may be successfully used as a drug delivery system for bisquaternary oximes and improve central nervous system targeting. The main aim of these studies was to elucidate the relationship between cucurbit[7]uril, oxime K027, atropine, and paraoxon to define potential risks or advantages of this delivery system in a complex in vivo system. For this reason, in silico (molecular docking combined with umbrella sampling simulation) and in vivo (UHPLC—pharmacokinetics, toxicokinetics; acetylcholinesterase reactivation and functional observatory battery) methods were used. Based on our results, cucurbit[7]urils affect multiple factors in organophosphates poisoning and its therapy by (i) scavenging paraoxon and preventing free fraction of this toxin from entering the brain, (ii) enhancing the availability of atropine in the central nervous system and by (iii) increasing oxime passage into the brain. In conclusion, using cucurbit[7]urils with oximes might positively impact the overall treatment effectiveness and the benefits can outweigh the potential risks.     

DM1 Transgenic Mice Exhibit Abnormal Neurotransmitter Homeostasis and Synaptic Plasticity in Association with RNA Foci and Mis-Splicing in the Hippocampus

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease mediated by a toxic gain of function of mutant RNAs. The neuropsychological manifestations affect multiple domains of cognition and behavior, but their etiology remains elusive. Transgenic DMSXL mice carry the DM1 mutation, show behavioral abnormalities, and express low levels of GLT1, a critical regulator of glutamate concentration in the synaptic cleft. However, the impact of glutamate homeostasis on neurotransmission in DM1 remains unknown. We confirmed reduced glutamate uptake in the DMSXL hippocampus. Patch clamp recordings in hippocampal slices revealed increased amplitude of tonic glutamate currents in DMSXL CA1 pyramidal neurons and DG granule cells, likely mediated by higher levels of ambient glutamate. Unexpectedly, extracellular GABA levels and tonic current were also elevated in DMSXL mice. Finally, we found evidence of synaptic dysfunction in DMSXL mice, suggestive of abnormal short-term plasticity, illustrated by an altered LTP time course in DG and in CA1. Synaptic dysfunction was accompanied by RNA foci accumulation in localized areas of the hippocampus and by the mis-splicing of candidate genes with relevant functions in neurotransmission. Molecular and functional changes triggered by toxic RNA may induce synaptic abnormalities in restricted brain areas that favor neuronal dysfunction.     

叶黄素对母鼠乳腺分泌IgA的促进作用

为明确叶黄素对母鼠乳腺分泌IgA能力的影响,本实验对妊娠母鼠添加饲喂叶黄素,检测母鼠血清中IgG、IgA和IgM含量变化,母鼠乳腺组织中IgA mRNA表达量和IgA抗体分泌细胞数量变化,以及仔鼠胃内容物中IgA浓度变化。结果表明：在妊娠21 d和分娩后7 d时,实验组血清中IgG和IgA浓度显著高于正常对照组（P＜0.05）,IgM含量虽有升高,与正常对照组相比,无显著性差异（P＞0.05）;乳腺组织中IgA mRNA表达量无显著变化（P＞0.05）;分娩后14 d IgA抗体分泌细胞数量显著增加（P＜0.05）;仔鼠胃内容物中IgA含量极显著高于对照组（P＜0.01）。说明给妊娠母鼠添加饲喂叶黄素可以增强乳腺分泌IgA的能力。     

基于16S rRNA技术分析当归超微粉对小鼠肠道菌群多样性的影响

研究不同剂量当归超微粉对小鼠肠道菌群多样性、肠道生理结构的作用。将C57BL/6小鼠分为当归超微粉高剂量组(2.0g/kg)、中剂量组(1.0 g/kg)、低剂量组(0.5 g/kg)、传统饮片组(2.0 g/kg)、传统粉末组(2.0 g/kg)和空白组,灌胃给药15 d,记录体重,在灌喂第15 d时无菌取结肠内容物,扩增肠道菌群16S rRNA基因的V3+V4区,采用高通量测序分析β多样性和α多样性、分析门水平和属水平的物种组成,HE染色法观察胃肠道病理变化。结果显示,C57BL/6小鼠体重正常生长,当归超微粉饮片高剂量组的对小鼠肠道微生物的疣微菌门(Verrucomicrobia)含量显著增加(p<0.05),放线菌门(Actinobacteria)含量减少(p<0.01)。另外,当归超微粉饮片中剂量组拟杆菌门(Bacteroidetes)含量降低最为显著,厚壁菌门(Firmicutes)增加最为显著;在属水平中剂量Faecalibaculum增加最明显(p<0.05),故超微粉的中剂量为最佳实验剂量。各处理组小鼠胃肠道无病理变化。本实验可为当归以超微粉的使用方式,对人体保健或治疗使用的科学用量和安全性,提供理论依据。另外,可为以肠道菌群相关的有效性提供技术支持。     

Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current,If, in Adult Mouse Heart Primary Pacemaker Myocytes

A number of distinct electrophysiological mechanisms that modulate the myogenic spontaneous pacemaker activity in the sinoatrial node (SAN) of the mammalian heart have been investigated extensively. There is agreement that several (3 or 4) different transmembrane ionic current changes (referred to as the voltage clock) are involved; and that the resulting net current interacts with direct and indirect effects of changes in intracellular Ca²⁺ (the calcium clock). However, significant uncertainties, and important knowledge gaps, remain concerning the functional roles in SAN spontaneous pacing of many of the individual ion channel- or exchanger-mediated transmembrane current changes. We report results from patch clamp studies and mathematical modeling of the hyperpolarization-activated current, I_f, in the generation/modulation of the diastolic depolarization, or pacemaker potential, produced by individual myocytes that were enzymatically isolated from the adult mouse sinoatrial node (SAN). Amphotericin-mediated patch microelectrode recordings at 35 °C were made under control conditions and in the presence of 5 or 10 nM isoproterenol (ISO). These sets of results were complemented and integrated with mathematical modeling of the current changes that take place in the range of membrane potentials (−70 to −50 mV), which corresponds to the ‘pacemaker depolarization’ in the adult mouse SAN. Our results reveal a very small, but functionally important, approximately steady-state or time-independent current generated by residual activation of I_f channels that are expressed in these pacemaker myocytes. Recordings of the pacemaker depolarization and action potential, combined with measurements of changes in I_f, and the well-known increases in the L-type Ca²⁺ current, I_CaL, demonstrated that I_CaL activation, is essential for myogenic pacing. Moreover, after being enhanced (approximately 3-fold) by 5 or 10 nM ISO, I_CaL contributes significantly to the positive chronotropic effect. Our mathematical model has been developed in an attempt to better understand the underlying mechanisms for the pacemaker depolarization and action potential in adult mouse SAN myocytes. After being updated with our new experimental data describing I_f, our simulations reveal a novel functional component of I_f in adult mouse SAN. Computational work carried out with this model also confirms that in the presence of ISO the residual activation of I_f and opening of I_CaL channels combine to generate a net current change during the slow diastolic depolarization phase that is essential for the observed accelerated pacemaking rate of these SAN myocytes.     

The Ameliorative Effect of Dexamethasone on the Development of Autoimmune Lung Injury and Mediastinal Fat-Associated Lymphoid Clusters in an Autoimmune Disease Mouse Model

In our previous study, we revealed the ameliorative therapeutic effect of dexamethasone (Dex) for Lupus nephritis lesions in the MRL/MpJ-Fas ^lpr/lpr (Lpr) mouse model. The female Lpr mice developed a greater number of mediastinal fat-associated lymphoid clusters (MFALCs) and inflammatory lung lesions compared to the male mice. However, the effect of Dex, an immunosuppressive drug, on both lung lesions and the development of MFALCs in Lpr mice has not been identified yet. Therefore, in this study, we compared the development of lung lesions and MFALCs in female Lpr mice that received either saline (saline group “SG”) or dexamethasone (dexamethasone group “DG”) in drinking water as a daily dose along with weekly intraperitoneal injections for 10 weeks. Compared to the SG group, the DG group showed a significant reduction in the levels of serum anti-dsDNA antibodies, the size of MFALCs, the degree of lung injury, the area of high endothelial venules (HEVs), and the number of proliferating and immune cells in both MFALCs and the lungs. A significant positive correlation was observed between the size of MFALCs and the cellular aggregation in the lungs of Lpr mice. Therefore, this study confirmed the ameliorative effect of Dex on the development of lung injury and MFALCs via their regressive effect on both immune cells’ proliferative activity and the development of HEVs. Furthermore, the reprogramming of MFALCs by targeting immune cells and HEVs may provide a therapeutic strategy for autoimmune-disease-associated lung injury.