A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences.

A panel of monoclonal antibodies (mAbs) specific for the nucleocapsid (N) protein or the glycoprotein Gc of Schmallenberg virus (SBV), a novel member of the Simbu serogroup (genus Orthobunyavirus, family Bunyaviridae), was produced and used to analyze antigenic differences among members of this serogroup. Reactivity with various SBV-isolates and other Simbu serogroup viruses was assessed by an indirect immunofluorescence test and by immunoblotting. The Gc-specific mAbs detected different SBV isolates as well as two closely related members of the Simbu serogroup. In addition, one mAb showed a highly specific reactivity with the homologous SBV strain only. Based on their differing reactivity with different SBV-strains, these antibodies represent a valuable novel tool to rapidly determine the phenotype of new SBV isolates. In contrast, the N-specific mAbs showed a broad reactivity spectrum and detected not only all the tested SBV-isolates, but also several other viruses of the Simbu serogroup. One out of these mAbs even recognized all of the tested Simbu serogroup viruses in the indirect immunofluorescence assay. In order to further characterize the N-specific antibodies, PepScan analysis was performed and a specific epitope could be identified. In summary, the newly generated mAbs showed differing pan-Simbu virus-, pan-SBV- as well as SBV-isolate-specific reactivity patterns. Thus, they represent valuable tools for the development of novel antigen and antibody detection systems either specific for SBV or, in a broader approach, for the pan-Simbu serogroup diagnostics.

Amyloid precursor protein knockout mice show age-dependent deficits in passive avoidance learning.

Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD), but its role in cognition has been relatively little studied. APP knockout (KO) animals have been described previously and show deficits in grip strength, reduced locomotor activity and impaired learning and memory in a conditioned avoidance test and the Morris water-maze. In order to further investigate the in vivo function of APP and its proteolytic derivatives, we tested APP KO mice and age-matched wild type controls at two different ages, 3 and 8 months, in a range of behavioural tests measuring neuromuscular, locomotor and cognitive functions. These tests included the acquisition of a passive avoidance response as a measure of long-term memory of an aversive experience, and spontaneous alternation in a Y-maze, regarded as a measure of spatial short-term memory. The absence of APP expression in APP KO mice was confirmed at the protein level using hippocampal tissue in Western blotting. APP KO mice displayed deficits in forelimb grip strength and locomotor activity in agreement with previous studies. In the Y-maze test used for spontaneous alternation behaviour, APP KO animals did not exhibit reduced alternation rates. On the other hand, in the passive avoidance test, APP KO mice showed an age-related deficit in retention of memory for an aversive experience. The results suggest that APP and/or its proteolytic derivatives may play a role in long-term memory in adult brain and/or may be required during the development and maintenance of neuronal networks involved in this type of memory.

Increased exploratory activity of APP23 mice in a novel environment is reversed by siRNA.

Genetic abnormalities in amyloid precursor protein (APP) are associated with Down's syndrome and familial Alzheimer's disease where hallmark plaques contain A beta peptides derived from APP. Both APP and its derivatives are implicated in neurodegenerative processes and may play important physiological and pathophysiological roles in synaptic function. Here, we show that young APP23 transgenic mice overexpressing human APP with the Swedish double mutation display altered novelty seeking behavior before the age of plaque onset. Using short interfering RNA (siRNA) targeted against APP, we investigate the direct contribution of APP and its derivatives to this behavioral deficit. After validating siRNAs targeting human APP in vitro, siRNAs were infused directly into the brain of APP23 mice for 2 weeks. Behavioral analysis shows that infusion of siRNA targeted against APP completely reverses increased exploratory activity in APP23 mice. Collectively, these data suggest that excessive APP and/or its derivatives, causes a hyperactive phenotype in APP23 mice when placed in a novel environment, which is fully reversible and not linked to plaque deposits.

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.

Unraveling in vivo functions of amyloid precursor protein: insights from knockout and knockdown studies.

The amyloid precursor protein (APP) is a widely expressed transmembrane protein that is cleaved to generate Abeta peptides in the central nervous system and is a key player in the pathogenesis of Alzheimer's disease. The precise biological functions of APP still remain unclear although various roles have been proposed. While a commonly accepted model argues that Abeta peptides are the cause of onset and early pathogenesis of Alzheimer's disease, recent discussions challenge this 'Abeta hypothesis' and suggest a direct role for APP in this neurodegenerative disease. Loss-of-function studies are an efficient way to elucidate the role of proteins and concurrently a variety of in vitro and in vivo studies has been performed for APP where protein levels have been downregulated and functional consequences monitored. Complete disruption of APP gene expression has been achieved by the generation of APP knockout animal models. Further knockdown studies using antisense and RNA interference have allowed scientists to reduce APP expression levels and have opened new avenues to explore the physiological roles of APP. In the present review, we focus on knockout and knockdown approaches that have provided insights into the physiological functions of APP and discuss their advantages and drawbacks.