Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease

<p>Abstract</p> <p>Background</p> <p>Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.</p> <p>Results</p> <p>For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.</p> <p>Conclusions</p> <p>The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.</p

Genetic inactivation of the vesicular glutamate transporter 2 (VGLUT2) in the mouse: What have we learnt about functional glutamatergic neurotransmission?

During the past decade, three proteins that possess the capability of packaging glutamate into presynaptic vesicles have been identified and characterized. These three vesicular glutamate transporters, VGLUT1–3, are encoded by solute carrier genes Slc17a6–8. VGLUT1 (Slc17a7) and VGLUT2 (Slc17a6) are expressed in glutamatergic neurons, while VGLUT3 (Slc17a8) is expressed in neurons classically defined by their use of another transmitter, such as acetylcholine and serotonin. As glutamate is both a ubiquitous amino acid and the most abundant neurotransmitter in the adult central nervous system, the discovery of the VGLUTs made it possible for the first time to identify and specifically target glutamatergic neurons. By molecular cloning techniques, different VGLUT isoforms have been genetically targeted in mice, creating models with alterations in their glutamatergic signalling. Glutamate signalling is essential for life, and its excitatory function is involved in almost every neuronal circuit. The importance of glutamatergic signalling was very obvious when studying full knockout models of both VGLUT1 and VGLUT2, none of which were compatible with normal life. While VGLUT1 full knockout mice die after weaning, VGLUT2 full knockout mice die immediately after birth. Many neurological diseases have been associated with altered glutamatergic signalling in different brain regions, which is why conditional knockout mice with abolished VGLUT-mediated signalling only in specific circuits may prove helpful in understanding molecular mechanisms behind such pathologies. We review the recent studies in which mouse genetics have been used to characterize the functional role of VGLUT2 in the central nervous system

Soluble amyloid-β aggregates in Alzheimer’s disease

Soluble oligomeric aggregates of the amyloid-β (Aβ) peptide are suggested to initiate Alzheimer's disease (AD), leading to impaired synapse signalling, widespread neuronal death and loss of cognitive functions. These aggregates seem tightly linked to disease progression, and have therefore gained much attention as potential novel disease markers. In this thesis soluble oligomeric Aβ aggregates in general, and the Aβ protofibril species in particular, have been investigated with the aim to quantify and determine their role in AD pathogenesis. Sandwich-ELISAs specifically measuring Aβ42 peptides are widely used both in AD research and as complements for clinical diagnosis. Here it was demonstrated that presence of soluble Aβ aggregates disturbs such analyses, making it difficult to interpret the results. This discovery was made through analyses of samples from cell- and mouse models carrying the AD causing 'Arctic' APP mutation. When analyzed by ELISA, Aβ42 levels were reduced in Arctic samples, in contrast to levels measured by denaturing SDS-PAGE Western blot. The same divergence in Aβ42-levels between analyses was observed in CSF samples from Down syndrome infants. The discrepancy between methods was hypothesized to be due to presence of soluble Aβ aggregates leading to impaired ELISA detection caused by epitope masking. This was confirmed by developing a protofibril specific ELISA, by which samples from Arctic cell- and mouse models were demonstrated to have enhanced Aβ protofibril levels. AD patients have reduced ELISA-measured Aβ42-levels in CSF compared to healthy controls. To test if this reduction was due to oligomeric Aβ species present in AD CSF, Aβ42-levels were analyzed under both denaturing and non-denaturing conditions. These two measures were combined and an Aβ42 oligomer ratio established. Higher ratios were found in AD patients than healthy controls, implying that Aβ oligomers are present in CSF during Alzheimer pathogenesis. The observations from AD patients and young Down syndrome individuals suggest that Aβ42 oligomer formation is an early mechanism of AD pathogenesis, which potentially could be used as a biomarker to monitor disease development

Performance measurement systems as management control in R&amp;D organizations : A case study

Background: Management control systems (MCS) are used to control organizations and make employees behave and act in the desirable way. Performance measurement (PM) systems one type of MCS and are used to communicate company strategies throughout the organization, motivate the employees to work towards company goals, and measure the outcome. PM systems can be a powerful tool, but if used in the wrong way they can have adverse effects. Aim: This thesis focused on the use of PM systems for management control purposes in research and development (R&amp;D) organisations with the question: How can performance measurement systems be utilized in R&amp;D organizations? Method: The thesis is based on a literature study, complemented by a case study (metric analysis, survey and deep interviews) at a R&amp;D department. The department was investigated at two time points, in between which the PM system was re-designed. In the metric analysis, the performance targets of the PM system were categorized into quantitative-objective, quantitative-subjective and qualitative-subjective targets. Results: The results from the case study were in line with findings from the literature. At study point 2, when the PM system had been re-designed, the employees felt more involved in shaping and influencing the goals. Also the follow-up of the goals was experienced as more implemented at study point two. The types of measured targets had shifted from quantitative to qualitative, including soft values such as team spirit, at study point 2. However, the members did not feel that the goals motivated them at any time point. . Conclusion: In the literature review it was evident from the number of publications that there is a great interest in measuring R&amp;D performance, and that PM systems are an important tool to R&amp;D managers. Just as the company in this case study, each organization needs to analyze its own needs and adopt the PM system thereafter. Moreover, no system should be seen as static, instead it should be continuously evaluated and adjusted to make sure it measures what it is intended to measure and that it does not cause adverse effects on the organization

Performance measurement systems as management control in R&amp;D organizations : A case study

Background: Management control systems (MCS) are used to control organizations and make employees behave and act in the desirable way. Performance measurement (PM) systems one type of MCS and are used to communicate company strategies throughout the organization, motivate the employees to work towards company goals, and measure the outcome. PM systems can be a powerful tool, but if used in the wrong way they can have adverse effects. Aim: This thesis focused on the use of PM systems for management control purposes in research and development (R&amp;D) organisations with the question: How can performance measurement systems be utilized in R&amp;D organizations? Method: The thesis is based on a literature study, complemented by a case study (metric analysis, survey and deep interviews) at a R&amp;D department. The department was investigated at two time points, in between which the PM system was re-designed. In the metric analysis, the performance targets of the PM system were categorized into quantitative-objective, quantitative-subjective and qualitative-subjective targets. Results: The results from the case study were in line with findings from the literature. At study point 2, when the PM system had been re-designed, the employees felt more involved in shaping and influencing the goals. Also the follow-up of the goals was experienced as more implemented at study point two. The types of measured targets had shifted from quantitative to qualitative, including soft values such as team spirit, at study point 2. However, the members did not feel that the goals motivated them at any time point. . Conclusion: In the literature review it was evident from the number of publications that there is a great interest in measuring R&amp;D performance, and that PM systems are an important tool to R&amp;D managers. Just as the company in this case study, each organization needs to analyze its own needs and adopt the PM system thereafter. Moreover, no system should be seen as static, instead it should be continuously evaluated and adjusted to make sure it measures what it is intended to measure and that it does not cause adverse effects on the organization

High frequency of IgE sensitization towards kiwi seed storage proteins among peanut allergic individuals also reporting allergy to kiwi

Abstract Background IgE sensitization to storage proteins from nuts and seed is often related to severe allergic symptoms. There is a risk of immunological IgE cross-reactivity between storage proteins from different species. The potential clinical implication of such cross-reactivity is that allergens other than the known sensitizer can cause allergic symptoms. Previous studies have suggested that kiwi seed storage proteins may constitute hidden food allergens causing cross-reactive IgE-binding with peanut and other tree nut homologs, thereby mediating a potential risk of causing allergy symptoms among peanut ant tree nut allergic individuals. The objective of this study was to investigate the degree of sensitization towards kiwi fruit seed storage proteins in a cohort of peanut allergic individuals. Methods A cohort of 59 adolescents and adults with peanut allergy was studied, and self reported allergies to a number of additional foods were collected. Quantitative IgE measurements to seed storage proteins from kiwi and peanut were performed. Results In the cohort, 23 out of the 59 individuals were reporting kiwi fruit allergy (39%). The frequency of IgE sensitization to kiwi fruit and to any kiwi seed storage protein was higher among peanut allergic individuals also reporting kiwi fruit allergy (P = 0.0001 and P = 0.01). A positive relationship was found between IgE levels to 11S globulin (r = 0.65) and 7S globulin (r = 0.48) allergens from kiwi and peanut, but IgE levels to 2S albumin homologs did not correlate. Patients reporting kiwi fruit allergy also reported allergy to hazelnut (P = 0.015), soy (P < 0.0001), pea (P = 0.0002) and almond (P = 0.016) to a higher extent than peanut allergic individuals without kiwi allergy. Conclusions Thirty-nine percent of the peanut allergic patients in this cohort also reported kiwi fruit allergy, they displayed a higher degree of sensitization to kiwi storage proteins from both kiwi and peanut, and they also reported a higher extent of allergy to other nuts and legumes. On the molecular level, there was a correlation between IgE levels to 11S and 7S storage proteins from kiwi and peanut. Taken together, reported symptoms and serological findings to kiwi in this cohort of patients with concurrent allergy to peanut and kiwi fruit, could be explained by a combination of cross-reactivity between the 11S and 7S globulins and co-sensitization to the 2S albumin Act d 13

Increase in β-Amyloid Levels in Cerebrospinal Fluid of Children with Down Syndrome

Background: Individuals with Down syndrome (DS) invariably develop Alzheimer’s disease (AD) during their life span. It is therefore of importance to study young DS patients when trying to elucidate early events in AD pathogenesis. Aim: To investigate how levels of different amyloid- _ (A _ ) peptides, as well as tau and phosphorylated tau, in cerebrospinal fluid (CSF) from children with DS change over time. The first CSF sample was taken at 8 months and the following two samples at 20–40 and 54 months of age. Results: Individual levels of the A _ peptides, as well as total A _ levels in CSF increased over time when measured with Western blot. Tau in CSF decreased whereas there was no change in levels of phosphorylated tau over time. Conclusion: The increasing levels of A _ in CSF during early childhood of DS patients observed in this study are probably due to the trisomy of the A _ precursor APP, which leads to an overproduction of A _ . Despite the increased CSF concentrations of A _ , there were no signs of an AD-indicating tau pattern in CSF, since the levels of total tau decreased and phosphorylated tau remained unchanged. This observation further strengthens the theory of A _ pathology preceding tau pathology in AD

Large aggregates are the major soluble Aβ species in AD brain fractionated with density gradient ultracentrifugation.

Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80-500 kDa synthetic Aβ protofibrils and were equally detected with mAb158