Mouse Models for Studying the Formation and Propagation of Prions

Prions are self-propagating protein conformers that cause a variety of neurodegenerative disorders in humans and animals. Mouse models have played key roles in deciphering the biology of prions and in assessing candidate therapeutics. The development of transgenic mice that form prions spontaneously in the brain has advanced our understanding of sporadic and genetic prion diseases. Furthermore, the realization that many proteins can become prions has necessitated the development of mouse models for assessing the potential transmissibility of common neurodegenerative diseases. As the universe of prion diseases continues to expand, mouse models will remain crucial for interrogating these devastating illnesses.     

Evaluating Prion Models Based on Comprehensive Mutation Data of Mouse PrP

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Mouse Models of Human Phenylketonuria

Phenylketonuria (PKU) results from a deficiency in phenylalanine hydroxylase, the enzyme catalyzing the conversion of phenylalanine (PHE) to tyrosine. Although this inborn error of metabolism was among the first in humans to be understood biochemically and genetically, little is known of the mechanism(s) involved in the pathology of PKU. We have combined mouse germline mutagenesis with screens for hyperphenylalaninemia to isolate three mutants deficient in phenylalanine hydroxylase (PAH) activity and cross-reactive protein. Two of these have reduced PAH mRNA and display characteristics of untreated human PKU patients. A low PHE diet partially reverses these abnormalities. Our success in using high frequency random germline point mutagenesis to obtain appropriate disease models illustrates how such mutagenesis can complement the emergent power of targeted mutagenesis in the mouse. The mutants now can be used as models in studying both maternal PKU and somatic gene therapy.     

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Mouse Models of Diet-Induced Nonalcoholic Steatohepatitis Reproduce the Heterogeneity of the Human Disease

MethodsMice were fed standard chow, MCD diet for 8 weeks, or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. Liver pathology and metabolic profile were compared.ResultsThe metabolic profile associated with human NASH was better mimicked by Western diet. Although hepatic steatosis (i.e., triglyceride accumulation) was also more severe, liver non-esterified fatty acid content was lower than in the MCD diet group. NASH was also less severe and less reproducible in the Western diet model, as evidenced by less liver cell death/apoptosis, inflammation, ductular reaction, and fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also less robust in the Western diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation.ConclusionFeeding mice a Western diet models metabolic perturbations that are common in humans with mild NASH, whereas administration of a MCD diet better models the pathobiological mechanisms that cause human NAFLD to progress to advanced NASH.     

人类疾病模型小鼠的标准化维持方法研究

目的研究人类疾病模型小鼠的可遗传生物学特性,建立品系的标准化维持方法。方法选用FVB TgN、MRL Mpj和SAMP1Ka三个疾病模型小鼠品系作为代表性实验对象,通过测定生长曲线、RAPD同工、酶电泳、行为学试验等方法,找出三个品系相对于各自对照品系的不同特征,并建立了标准化检测指标作为维持方法的依据。结果MRL Mpj的生长曲线相对于对照品系有明显的统计学差异;FVB TgN、MRL Mpj、SAMP1Ka等三个品系的RAPD图谱在阳性引物及扩增出的条带方面均不一致;同工酶电泳的结果表明不同品系的个体之间表型不完全相同;行为学试验更从多方面直观地显示了各品系的不同特征。结论人类疾病模型小鼠除了用常规检测方法外,还应建立各品系在生长曲线、RAPD、同工酶电泳、行为学试验等方面的特殊检测方法,及时检测模型小鼠品系的独特性状并作为保种依据,以避免遗传漂变的发生。     

Studying Human Pathogens in Animal Models: Fine Tuning the Humanized Mouse

Humanized mice are crucial tools for studying human pathogens in systemic situations. An animal model of human coronavirus infectious disease has been generated by gene transfer of the human receptor for virus-cell interaction (aminopeptidase N, APN, CD13) into mice. We showed that in vitro and in vivo infections across the species barrier differ in their requirements. Transgenic cells were susceptible to human coronavirus HCoV-229E infection demonstrating the requirement of hAPN for viral cell entry. Transgenic mice, however, could not be infected suggesting additional requirements for in vivo virus susceptibility. Crossing hAPN transgenic mice with interferon unresponsive Stat1^−/− mice resulted in markedly enhanced virus replication in vitro but did not result in detectable virus replication in vivo. Adaptation of the human virus to murine cells led to successful infection of the humanized transgenic mice. Future genetic engineering approaches are suggested to provide animal models for the better understanding of human infectious diseases.     

The Pathological Phenotypes of Human TDP-43 Transgenic Mouse Models Are Independent of Downregulation of Mouse Tdp-43

Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-shTardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice.     

Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment

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裸鼠肿瘤动物模型VEGF受体表达及其意义

目的　通过免疫组织化学染色了解flt 1与flk 1 KDR(VEGF的两个高亲和受体 )在人肿瘤细胞皮下接种肿瘤动物模型的血管内皮细胞与肿瘤细胞中的表达。方法　取荷瘤裸鼠皮下接种瘤块 ,漂洗、固定、石蜡连续切片 ,进行两种受体相应免疫组化检测。结果　在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中flt 1的阳性率大部分为强阳性或中阳性 ,而只有在荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中flt 1的阳性率为弱阳性 ,在荷人卵巢癌SKOv3裸鼠的肿瘤细胞中flt 1的表达为阴性。相比较而言 ,在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中KDR的阳性率大部分为中阳性或弱阳性 ,并且在荷人肝癌SMMC 772 1裸鼠 ,荷人胃腺癌SPC A1裸鼠 ,荷人高转移肝癌移植瘤裸鼠 ,荷人卵巢癌SKOv3裸鼠的肿瘤细胞中 ,荷人宫颈癌移植瘤裸鼠和荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中 ,KDR表达为阴性。结论　VEGF受体共同表达于肿瘤血管内皮细胞与肿瘤细胞 ,提示了VEGF与VEGF受体结合作用在肿瘤演化中的重要性 ,为靶向于VEGF受体的基因治疗策略选择裸鼠动物模型提供了参考依据     

Validation of Transgenic Mammary Cancer Models: Goals of the NCI Mouse Models of Human Cancer Consortium and the Mammary Cancer CD-ROM

Transgenic Research -     

人PrP基因体外诱导细胞凋亡的初步研究

朊蛋白为可传播性海绵样脑病的感染因子。将编码朊病毒白的ＰｒＰ标准及突变ＤＮＡ序列分别连接到真核表达载体ｐｃＤＮＡ３．１中,并分别转入中国仓鼠卵巢细胞（ＣＨＯ）。以过ＲＴ－ＰＣＲ、ＲＮＡｄｏｔ ｂｌｏｔ、Ｗｅｓｔｅｒｎ ｂｌｏｔ鉴定证实,得到了稳定表达人标准（ＣＨＯｓ）和终止密码突变ＰｒＰ基因（ＣＨＯｍ）的细胞系。对此细胞系进一步研究发现,带有标准人ＰｒＰ序列的ＣＨＯ细胞系的生长速度比其它对照细胞系     

Development of Functional Human NK Cells in an Immunodeficient Mouse Model with the Ability to Provide Protection against Tumor Challenge

Studies of human NK cells and their role in tumor suppression have largely been restricted to in vitro experiments which lack the complexity of whole organisms, or mouse models which differ significantly from humans. In this study we showed that, in contrast to C57BL/6 Rag2^−/−/γ_c ^−/− and NOD/Scid mice, newborn BALB/c Rag2^−/−/γ_c ^−/− mice can support the development of human NK cells and CD56+ T cells after intrahepatic injection with hematopoietic stem cells. The human CD56⁺ cells in BALB/c Rag2^−/−/γ_c ^−/− mice were able to produce IFN-γ in response to human IL-15 and polyI:C. NK cells from reconstituted Rag2^−/−/γ_c ^−/− mice were also able to kill and inhibit the growth of K562 cells in vitro and were able to produce IFN-γ in response to stimulation with K562 cells. In vivo, reconstituted Rag2^−/−/γ_c ^−/− mice had higher survival rates after K562 challenge compared to non-reconstituted Rag2^−/−/γ_c ^−/− mice and were able to control tumor burden in various organs. Reconstituted Rag2^−/−/γ_c ^−/− mice represent a model in which functional human NK and CD56+ T cells can develop from stem cells and can thus be used to study human disease in a more clinically relevant environment.     

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP^C into its pathogenic isoform PrP^{Sc 1}. PrP^C is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP^Sc forms detergent-insoluble aggregates and is partially resistant to PK^2-6. The conversion of PrP^C to PrP^Sc is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP^Sc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain⁷.Using a combination of biophysical and biochemical approaches, we identified insoluble PrP^C aggregates (designated iPrP^C) from uninfected mammalian brains and cultured neuronal cells^{8, 9}. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms¹⁰, and PK-treatment. The combination of these approaches isolates not only insoluble PrP^Sc and PrP^C aggregates but also soluble PrP^C oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrP^Sc from infected brains and iPrP^C from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrP^C are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrP^Sc that shares the immunoreactive behavior and fragmentation with iPrP^{C 11, 12}. Moreover, we recently demonstrated that iPrP^C is the main species that interacts with amyloid-β protein in Alzheimer disease¹³. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer''s disease¹³, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.     

Mouse Models for the Study of SARS-CoV-2 Infection

Mice are an invaluable resource for studying virus-induced disease. They are a small, genetically modifiable animal for which a large arsenal of genetic and immunologic tools is available for evaluation of pathogenesis and potential vaccines and therapeutics. SARS-CoV-2, the betacoronavirus responsible for the COVID-19 pandemic, does not naturally replicate in wild-type mice, due to structural differences between human and mouse ACE2, the primary receptor for SARS-CoV-2 entry into cells. However, several mouse strains have been developed that allow for SARS-CoV-2 replication and clinical disease. Two broad strategies have primarily been deployed for developing mouse strains susceptible to COVID-19-like disease: adding in the human ACE2 gene and adapting the virus to the mouse ACE2 receptor. Both approaches result in mice that develop several of the clinical and pathologic hallmarks of COVID-19, including acute respiratory distress syndrome and acute lung injury. In this review, we describe key acute pulmonary and extrapulmonary pathologic changes seen in COVID-19 patients that mouse models of SARS-CoV-2 infection ideally replicate, the essential development of mouse models for the study of Severe Acute Respiratory Syndrome and Middle Eastern Respiratory Syndrome and the basis of many of the models of COVID-19, and key clinical and pathologic features of currently available mouse models of SARS-CoV-2 infection.

Coronaviruses are widespread and infect several different species. In humans, coronaviruses historically cause primarily mild respiratory diseases, such as the common cold. However, in 2003 a novel coronavirus emerged, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), which caused severe respiratory disease with high mortality.¹⁸ Since then, 2 other highly pathogenic coronaviruses from the same betacoronavirus genus have emerged: Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV) in 2012, and most recently, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).^18,133,134 Spread of SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19), has resulted in a once-in-a-lifetime pandemic, and as of May 2021, more than 160 million cases and more than 3.3 million deaths have been reported worldwide.¹²³Much like SARS-CoV, SARS-CoV-2 has 4 structural proteins—spike (S), envelope (E), membrane (M), and nucleocapsid (N)—and 8 accessory proteins. The S protein includes a receptor-binding domain, which is essential for the virus to bind to and subsequently infect a host cell.¹²⁴ SARS-CoV and SARS-CoV-2 both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor and transmembrane protease serine 2 as a cofactor,^42,56,60 In contrast, the receptor for MERS-CoV is dipeptidyl peptidase 4 (DPP4).⁹¹ Because of amino acid changes in the S protein, SARS-CoV-2 binds ACE2 with a higher affinity than does SARS-CoV,¹²⁴ which may explain the greater human infectivity of SARS-CoV-2.^42,102 ACE2 is expressed throughout the body, allowing SARS-CoV-2 to potentially infect multiple organs, including the lung, heart, kidney, liver, intestines, and brain. Importantly, ACE2 is expressed on the apical surfaces of epithelial cells in these organs, permitting infection from direct viral contact with those cells.³⁷ As its name suggests, ACE2 is a critical component of the renin–angiotensin cascade, limiting vasoconstriction and promoting vasodilation by converting angiotensin II to angiotensin 1–7. ACE2 in the lung is hypothesized to reduce lung inflammation; SARS-CoV and SARS-CoV-2 potentially could exacerbate lung inflammation by altering this pathway.⁹⁸A thorough understanding of disease pathogenesis and any potential vaccines or therapeutics for any emerging pathogen is facilitated by studying animal models. For example, the first FDA-approved treatment for COVID-19 was remdesivir. This drug initially was granted emergency-use authorization for COVID-19 patients in part because of demonstrated therapeutic efficacy against MERS-CoV infection in a mouse model.¹⁰³ An ideal animal model of COVID-19 captures the wide spectrum of disease phenotypes attributed to this multifaceted disease, including organ-specific pathology and systemic changes such as hypercoagulation and cytokine storms. Animal models rarely capture every aspect of human disease, requiring the use of multiple models in order to replicate the numerous features of viral infection and disease and thereby build a comprehensive picture of what happens in human patients.SARS-CoV-2 naturally infects numerous animal species, including mink on farms, big cats in zoos and sanctuaries, and domestic dogs and cats.^{28,36,68,72,81,87,104,107} Although mink are particularly vulnerable to severe SARS-CoV-2 disease and can transmit virus to humans, they are rarely used in research studies.^68,81,87 Mice, hamsters, ferrets, and nonhuman primates are more widely used as experimental models of SARS-CoV-2 infection.⁶⁸ Hamsters replicate human disease well, are small, and are widely available, making them an attractive choice.^12,68,105 However, hamsters are less commonly used in research than are mice, and the availability of strains and reagents necessary for studying genetic and immune drivers of disease is limited for hamsters. Ferrets are frequently used as models for respiratory viruses, particularly influenza, primarily because they disperse and are susceptible to these viruses via airborne droplets, whereas rodents do not. However, ferrets do not develop severe symptoms or generate high virus titers in the lungs after SARS-CoV-2 infection.^52,68,95,104 NHPs have similar immune responses to humans and are invaluable for safety studies for preclinical trials but are costly to use and require significant infrastructure to maintain studies.^66,68,130 Mice provide a valuable model because they are widely available and relatively inexpensive, and a large arsenal of genetic and immunologic tools is available for mice. The use of mice to study SARS-CoV-2 infection provides the potential for a broader understanding of several different aspects of this complex disease.     

LDLR Expression and Localization Are Altered in Mouse and Human Cell Culture Models of Alzheimer's Disease

Background

Alzheimer''s disease (AD) is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the ε-4 allele of apolipoprotein E (apoE), the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR) has the highest affinity for apoE and plays an important role in brain cholesterol metabolism.

Methodology/Principal Findings

Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Aβ-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of γ- and α-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network.

Conclusions/Significance

These data suggest that increased APP expression and Aβ exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.