Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein‐specific antibodies

Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age‐related and brain region‐specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP‐transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP‐transgenic mouse and one APP‐transgenic rat model. We observed remarkable differences in expression levels and brain region‐specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP‐transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.     

诺如病毒感染宿主免疫应答机制及疫苗研究进展

诺如病毒是引起人类急性胃肠炎的重要食源性病原之一。由于体外复制系统和感染模型的缺乏,研究人员对其宿主保护性免疫的理解始终有限,导致控制病毒感染方面的研究也受到较大阻碍。近年来,随着病毒衣壳蛋白外源表达、替代病毒的使用、志愿者实验的开展,尤其是细胞培养模型的突破,使得体液免疫和细胞免疫的研究取得较大进展。因此,本文针对诺如病毒感染宿主的先天性免疫、体液免疫和细胞免疫应答机制等进行了综述,并对后续其在诺如病毒候选疫苗研制等领域的应用进行了展望。     

Zika virus non-structural protein 4B interacts with DHCR7 to facilitate viral infection

Zika virus (ZIKV) evolves non-structural proteins to evade immune response and ensure efficient replication in the host cells. Cholesterol metabolic enzyme 7-dehydrocholesterol reductase (DHCR7) was recently reported to impact innate immune responses in ZIKV infection. However, the vital non-structural protein and mechanisms involved in DHCR7-mediated viral evasion are not well elucidated. In this study, we demonstrated that ZIKV infection facilitated DHCR7 expression. Notably, the upregulated DHCR7 in turn facilitated ZIKV infection and blocking DHCR7 suppressed ZIKV infection. Mechanically, ZIKV non-structural protein 4B (NS4B) interacted with DHCR7 to induce DHCR7 expression. Moreover, DHCR7 inhibited TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) phosphorylation, which resulted in the reduction of interferon-beta (IFN-β) and interferon-stimulated genes (ISGs) productions. Therefore, we propose that ZIKV NS4B binds to DHCR7 to repress TBK1 and IRF3 activation, which in turn inhibits IFN-β and ISGs, and thereby facilitating ZIKV evasion. This study broadens the insights on how viral non-structural proteins antagonize innate immunity to facilitate viral infection via cholesterol metabolic enzymes and intermediates.     

H102对APP转基因小鼠脑内淀粉样蛋白和淀粉样蛋白前体蛋白表达的影响

目的:研究H102对APP695转基因模型小鼠脑内淀粉样蛋白和淀粉样蛋白前体蛋白表达的影响方法:9月龄转基因小鼠随机分为模型组和药物注射组,正常对照组采用月龄和性别与之相匹配的C57BL/6J小鼠。药物注射组给予侧脑室注射H102,每只每次3μl,连续10d;模型组和正常对照组给予等体积NS。应用免疫组织化学结合刚果红组织学染色,普通光学显微镜观察海马和颞叶皮层蛋白表达的变化。免疫印迹法检测小鼠大脑皮层APP蛋白的表达。结果:Aβ和APP免疫组化染色结果显示对照组海马CA1区神经元胞浆着色呈阴性或弱阳性,模型组较对照组阳性细胞增多,表达增强,胞浆着色明显加深。药物注射组同模型组相比,胞浆着色变淡,表达减弱。刚果红染色观察转基因小鼠模型组和H102注射组大脑颞叶皮层和海马的淀粉样斑块,可见H102注射组淀粉样斑块数较模型组明显减少。正常对照组未见阳性淀粉样斑块。免疫印迹检测显示模型组APP蛋白表达明显增加,给药组与模型组相比具有统计学意义。结论:APP695转基因小鼠大脑CA1区Aβ蛋白和APP蛋白表达增加,H102能够明显抑制该转基因小鼠Aβ蛋白和APP蛋白表达。     

Growth hormone attenuates the brain damage caused by ZIKV infection in mice

As a member of vector-borne viruses, Zika virus (ZIKV) can cause microcephaly and various neurological symptoms in newborns. Previously, we found that ZIKV could infect hypothalamus, causing a decrease in growth hormone (GH) secretion, growth delay and deficits in learning and memory in suckling mice. Early administration of GH can improve the cognitive function of the mice. Therefore, in this study we further investigated the mechanism underlying the protective role of GH in ZIKV infection in suckling mice. Our results showed that GH could effectively reduce brain damage caused by ZIKV infection via reducing cell apoptosis and inflammatory response rather than inhibiting viral replication. Our results provide important evidences not only for understanding the mechanism underlying ZIKV-associated neurological symptoms but also for the treatment of ZIKV infection.     

Xanthenedione (and intermediates involved in their synthesis) inhibit Zika virus migration to the central nervous system in murine neonatal models

Zika Virus (ZIKV), an arbovirus that belongs to the Flaviviridae family, has become a global concern since its outbreak in the Americas in 2015. With symptoms similar to other Flavivirus as Dengue and Yellow Fever viruses, infections by ZIKV have also been related to several neurological complications such as microcephaly in newborns and Guillain-Barre syndrome. Considering the high prevalence of ZIKV infection in certain areas, the risks that the virus poses to fetal brain development, and the fact that there is no vaccine or specific prophylaxis available, an effective treatment capable of preventing the infection is of potential interest. Therefore, in the present investigation, the antiviral activity on ZIKV of a group of xanthenodiones and intermediate ketones involved in their synthesis was evaluated for the first time. It was found that the compound 2-(2,6-dichlorobenzylidene)cyclohexane-1,3-dione 27 was able to completely inhibit the viral infection of Vero cells as well as to significantly reduce viral load in the brains of newborn Swiss mice. These effects are related to a direct interaction of the compound with the viral particle, blocking the viral adsorption.     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Protective Effects of Caffeine on Chikungunya and Zika Virus Infections: An in Vitro and in Silico Study

Infection by viruses Chikungunya (CHIKV) and Zika (ZIKV) continue to be serious problems in tropical and subtropical areas of the world. Here, we evaluated the antiviral and virucidal activity of caffeine against CHIKV and ZIKV in Vero, A549, and Huh-7 cell lines. Results showed that caffeine displays antiviral properties against both viruses. By pre-and post-infection treatment, caffeine significantly inhibited CHIKV and ZIKV replication in a dose-dependent manner. Furthermore, caffeine showed a virucidal effect against ZIKV. Molecular docking suggests the possible binding of caffeine with envelope protein and RNA-dependent RNA polymerase of CHIKV and ZIKV. This is the first study that showed an antiviral effect of caffeine against CHIKV and ZIKV. Although further studies are needed to better understand the mechanism of caffeine-mediated repression of viral replication, caffeine appears to be a promising compound that could be used for in vivo studies, perhaps in synergy with other compounds present in daily beverages.     

Amyloid precursor protein knockdown by siRNA impairs spontaneous alternation in adult mice

The cleavage-product of amyloid precursor protein (APP) constitutes the core component of plaques found in the brains of Alzheimer's disease (AD) patients. APP is ubiquitously expressed and its precise physiological functions remain unclear. This protein has been proposed to regulate synaptic function and processes underlying learning and memory. While APP knockout mice show behavioral impairments, these may occur due to early changes during development and/or due to abolition of APP function in adult. To investigate the acute effects of APP knockdown without involving developmental processes, APP expression was reduced using RNA interference in adult mouse brain. Small interfering RNAs (siRNAs) that down-regulated mouse APP protein levels (APP-siRNA) were identified using an APP plasmid-siRNA co-transfection assay in mouse NIH/3T3 fibroblast cells. Infusion of APP-siRNAs into the ventricular system for 2 weeks also down-regulated APP mRNA in mouse brain. Highest knockdown of APP mRNA levels was found in the CA2-CA3 regions of the hippocampus. Mice treated with the most active APP-siRNA showed a significant reduction in spontaneous alternation rate in the Y-maze, without effects on forelimb grip strength or locomotor activity. These data suggest that acute knockdown of APP in adult mouse brain impairs hippocampus-dependent spatial working memory.     

Crystal Structure of the Capsid Protein from Zika Virus

Recently, Zika virus (ZIKV) emerged as a global public health concern and is distinct from other flaviviruses in many aspects, for example, causing transplacental infection, fetal abnormalities and vector-independent transmission through body fluids in humans. The capsid (C) protein is a multifunctional protein, since it binds to viral RNA in the process of nucleocapsid assembly and plays important roles in virus infection processes by interacting with cellular proteins, modulating cellular metabolism, apoptosis and immune response. Here we solved the crystal structure of ZIKV C protein at a resolution of 1.9 Å. The ZIKV C protein structure contains four α helices with a long pre-α1 loop and forms dimers. The unique long pre-α1 loop in ZIKV C contributes to the tighter association of dimeric assembly and renders a divergent hydrophobic feature at the lipid bilayer interface in comparison with the known C structures of West Nile and dengue viruses. We reported the interaction between the ZIKV C protein and lipid droplets through confocal microscopy analysis. Substitutions of key amino acids in the pre-α1 loop of ZIKV C disrupted the interaction with lipid droplets, indicating that the loop is critical for membrane association. We also recognized that ZIKV C protein possesses broad binding capability to different nucleotide types, including single-stranded and double-stranded RNAs or DNAs. Furthermore, the highly positively charged interface, mainly formed by α4 helix, is proposed to be responsible for nucleotide binding. These findings will greatly enhance our understanding of ZIKV C protein, providing information for anti-ZIKV drug design targeting the C protein.     

The implication of SUMO in intrinsic and innate immunity

Since its discovery, SUMOylation has emerged as a key post-translational modification involved in the regulation of host-virus interactions. SUMOylation has been associated with the replication of a large number of viruses, either through the direct modification of viral proteins or through the modulation of cellular proteins implicated in antiviral defense. SUMO can affect protein function via covalent or non-covalent binding. There is growing evidence that SUMO regulates several host proteins involved in intrinsic and innate immunity, thereby contributing to the process governing interferon production during viral infection; as well as the interferon-activated Jak/STAT pathway. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed antiviral proteins (defined as restriction factors), which confer direct viral resistance through a variety of mechanisms. The aim of this review is to evaluate the role of SUMO in intrinsic and innate immunity; highlighting the involvement of the TRIM family proteins, with a specific focus on the mechanism through which SUMO affects i- interferon production upon viral infection, ii-interferon Jak/STAT signaling and biological responses, iii-the relationship between restriction factors and RNA viruses.     

Neuronal localization of the TNFα converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques

The tumor necrosis factor (TNF)‐α converting enzyme (TACE) can cleave the cell‐surface ectodomain of the amyloid‐β precursor protein (APP), thus decreasing the generation of amyloid‐β (Aβ) by cultured non‐neuronal cells. While the amyloidogenic processing of APP in neurons is linked to the pathogenesis of Alzheimer's disease (AD), the expression of TACE in neurons has not yet been examined. Thus, we assessed TACE expression in a series of neuronal and non‐neuronal cell types by Western blots. We found that TACE was present in neurons and was only faintly detectable in lysates of astrocytes, oligodendrocytes, and microglial cells. Immunohistochemical analysis was used to determine the cellular localization of TACE in the human brain, and its expression was detected in distinct neuronal populations, including pyramidal neurons of the cerebral cortex and granular cell layer neurons in the hippocampus. Very low levels of TACE were seen in the cerebellum, with Purkinje cells at the granular‐molecular boundary staining faintly. Because TACE was localized predominantly in areas of the brain that are affected by amyloid plaques in AD, we examined its expression in a series of AD brains. We found that AD and control brains showed similar levels of TACE staining, as well as similar patterns of TACE expression. By double labeling for Aβ plaques and TACE, we found that TACE‐positive neurons often colocalized with amyloid plaques in AD brains. These observations support a neuronal role for TACE and suggest a mechanism for its involvement in AD pathogenesis as an antagonist of Aβ formation. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 40–46, 2001     

Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

The global Zika virus (ZIKV) outbreak and its link to foetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant‐produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes. Furthermore, PzE can be purified to >90% homogeneity with a one‐step Ni²⁺ affinity chromatography process. PzE are found to be highly immunogenic, as two doses of PzE elicited both potent zE‐specific antibody and cellular immune responses in mice. The delivery of PzE with alum induced a mixed Th1/Th2 immune response, as the antigen‐specific IgG isotypes were a mixture of high levels of IgG1/IgG2c and splenocyte cultures from immunized mice secreted significant levels of IFN‐gamma, IL‐4 and IL‐6. Most importantly, the titres of zE‐specific and neutralizing antibodies exceeded the threshold that correlates with protective immunity against multiple strains of ZIKV. Thus, our results demonstrated the feasibility of plant‐produced ZIKV protein antigen as effective, safe and affordable vaccines against ZIKV.     

Down's Syndrome: Up-Regulation of β-Amyloid Protein Precursor and τ mRNAs and Their Defective Coordination

Abstract: Almost all patients >40 years of age with Down's syndrome (DS) develop the pathology characteristic of Alzheimer's disease: abundant β-amyloid plaques and neurofibrillary tangles. We have investigated the gene expression of β-amyloid protein precursor (APR) and τ in DS and age-matched control brains and found that levels of both mRNAs were significantly elevated in DS. Such up-regulation was not observed in two other neuronal proteins. A correlation between total APP and τ mRNA levels was also found in DS brain but distinct from the pattern observed in normal brain. Although a proportionality existed between APP-695 mRNA and three-repeat τ mRNA in DS, the proportionality between APP-751 mRNA and four-repeat τ mRNA, which is normally present, was not observed. Thus, DS brains are primarily characterized by the up-regulation of τ mRNA as well as APP mRNA and disruption of the coordinate expression between APP-751 and four-repeat τ.     

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons

J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.     

Rottlerin plays an antiviral role at early and late steps of Zika virus infection

Infection of Zika virus (ZIKV) may cause microcephaly and other neurological disorders, while no vaccines and drugs are available. Our study revealed that rottlerin confers a broad antiviral activity against several enveloped viruses, including ZIKV, vesicular stomatitis virus, and herpes simplex virus, but not against two naked viruses (enterovirus 71 and encephalomyocarditis virus). Rottlerin does not have a direct virucidal effect on the virions, and its antiviral effect is independent of its regulation on PKCδ or ATP. Both pretreatment and post-treatment of rottlerin effectively reduce the viral replication of ZIKV. The pretreatment of rottlerin disturbs the endocytosis of enveloped viruses, while the post-treatment of rottlerin acts at a late stage through disturbing the maturation of ZIKV. Importantly, administration of rottlerin in neonatal mice significantly decreased the ZIKV replication in vivo, and alleviated the neurological symptoms caused by ZIKV. Our work suggests that rottlerin exerts an antiviral activity at two distinct steps of viral infection, and can be potentially developed as a prophylactic and therapeutic agent.     

Ubiquilin-1 and protein quality control in Alzheimer disease

Single nucleotide polymorphisms in the ubiquilin-1 gene may confer risk for late-onset Alzheimer disease (AD). We have shown previously that ubiquilin-1 functions as a molecular chaperone for the amyloid precursor protein (APP) and that protein levels of ubiquilin-1 are decreased in the brains of AD patients. We have recently found that ubiquilin-1 regulates APP trafficking and subsequent secretase processing by stimulating non-degradative ubiquitination of a single lysine residue in the cytosolic domain of APP. Thus, ubiquilin-1 plays a central role in regulating APP biosynthesis, trafficking and ultimately toxicity. As ubiquilin-1 and other ubiquilin family members have now been implicated in the pathogenesis of numerous neurodegenerative diseases, these findings provide mechanistic insights into the central role of ubiquilin proteins in maintaining neuronal proteostasis.     

Different Gene Networks Are Disturbed by Zika Virus Infection in A Mouse Microcephaly Model

The association of Zika virus (ZIKV) infection with microcephaly has raised alarm worldwide. Their causal link has been confirmed in different animal models infected by ZIKV. However, the molecular mechanisms underlying ZIKV pathogenesis are far from clear. Hence, we performed global gene expression analysis of ZIKV-infected mouse brains to unveil the biological and molecular networks underpinning microcephaly. We found significant dysregulation of the sub-networks associated with brain development, immune response, cell death, microglial cell activation, and autophagy amongst others. We provided detailed analysis of the related complicated gene networks and the links between them. Additionally, we analyzed the signaling pathways that were likely to be involved. This report provides systemic insights into not only the pathogenesis, but also a path to the development of prophylactic and therapeutic strategies against ZIKV infection.     

Impact of prior Dengue immunity on Zika vaccine protection in rhesus macaques and mice

Pre-existing immunity to flaviviruses can influence the outcome of subsequent flavivirus infections. Therefore, it is critical to determine whether baseline DENV immunity may influence subsequent ZIKV infection and the protective efficacy of ZIKV vaccines. In this study, we investigated the impact of pre-existing DENV immunity induced by vaccination on ZIKV infection and the protective efficacy of an inactivated ZIKV vaccine. Rhesus macaques and mice inoculated with a live attenuated DENV vaccine developed neutralizing antibodies (NAbs) to multiple DENV serotypes but no cross-reactive NAbs responses to ZIKV. Animals with baseline DENV NAbs did not exhibit enhanced ZIKV infection and showed no overall reduction in ZIKV vaccine protection. Moreover, passive transfer of purified DENV-specific IgG from convalescent human donors did not augment ZIKV infection in STAT2 ^-/- and BALB/c mice. In summary, these results suggest that baseline DENV immunity induced by vaccination does not significantly enhance ZIKV infection or impair the protective efficacy of candidate ZIKV vaccines in these models. These data can help inform immunization strategies in regions of the world with multiple circulating pathogenic flaviviruses.