Ann-Sofi Johansson

Sensitive ELISA detection of amyloid-β protofibrils in biological samples

Amyloid‐β (Aβ) protofibrils are known intermediates of the in vitro Aβ aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Aβ protofibrils in Alzheimer’s disease (AD). To verify their in vivo relevance and to establish a quantitative Aβ protofibril immunoassay, Aβ conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Aβ protofibrils without interference from Aβ monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Aβ protofibrils in both cell and animal models, proving that Aβ protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Aβ protofibril levels in the Arctic‐Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Aβ protofibrils in AD and has the potential of becoming an important diagnostic assay.

Amyloid-β oligomers are inefficiently measured by enzyme-linked immunosorbent assay

Amyloid‐β (Aβ) peptide levels are widely measured by enzyme‐linked immunosorbent assay (ELISA) in Alzheimers disease research. Here, we show that oligomerization of Aβ results in underestimated Aβ ELISA levels. The implications are that comprehensive analysis of soluble Aβ requires either sample pretreatment at denaturing conditions or novel conformation‐dependent immunoassays. Our findings might be of relevance for many neurodegenerative disorders in which soluble protein aggregates are the main neurotoxic species. Ann Neurol 2005;58:147–150

Physiochemical characterization of the Alzheimer's disease‐related peptides Aβ1–42Arctic and Aβ1–42wt

The amyloid β peptide (Aβ) is crucial for the pathogenesis of Alzheimers disease. Aggregation of monomeric Aβ into insoluble amyloid fibrils proceeds through several soluble Aβ intermediates, including protofibrils, which are believed to be central in the disease process. The main reason for this is their implication in familial Alzheimers disease with the Arctic amyloid precursor protein mutation (E693G). This mutation gives rise to early onset Alzheimers disease, and synthetic Aβ1–40Arctic displays an enhanced rate of protofibril formation in vitro[Nilsberth C, Westlind‐Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Naslund J & Lannfelt L. (2001) Nat Neurosci4, 887–893]. To increase our understanding of the mechanisms involved in Aβ aggregation, especially Aβ monomer oligomerization into protofibrils and protofibril fibrillization into fibrils, the kinetics of Aβ1–42wt and Aβ1–42Arctic aggregation were examined under different physiochemical conditions, such as concentration, temperature, ionic strength and pH. We used size exclusion chromatography for this purpose, where monomers are separated from protofibrils, and fibrils are separated from protofibrils in a centrifugation step. The Arctic mutation significantly accelerated both Aβ1–42wt protofibril formation and protofibril fibrillization. In addition, we demonstrated that two distinct chemical processes – monomer oligomerization and protofibril fibrillization – were affected differently by changes in the micro‐environment and that the Arctic mutation alters the peptide response to such changes.

Docosahexaenoic acid stimulates non‐amyloidogenic APP processing resulting in reduced Aβ levels in cellular models of Alzheimer's disease

Epidemiological studies suggest that a high intake of polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), is associated with a reduced risk of Alzheimers disease. Here, we examined the effects of DHA on amyloid precursor protein (APP) processing in cellular models of Alzheimers disease by analysing levels of different APP fragments, including amyloid‐β (Aβ). DHA administration stimulated non‐amyloidogenic APP processing and reduced levels of Aβ, providing a mechanism for the reported beneficial effects of DHA in vivo. However, an increased level of APP intracellular domain was also observed, highlighting the need to increase our knowledge about the relevance of this fragment in Alzheimers disease pathogenesis. In conclusion, our results suggest that the proposed protective role of DHA in Alzheimers disease pathogenesis might be mediated by altered APP processing and Aβ production.

Docosahexaenoic acid stabilizes soluble amyloid-β protofibrils and sustains amyloid-β induced neurotoxicity in vitro

Enrichment of diet and culture media with the polyunsaturated fatty acid docosahexaenoic acid has been found to reduce the amyloid burden in mice and lower amyloid‐β (Aβ) levels in both mice and cultured cells. However, the direct interaction of polyunsaturated fatty acids, such as docosahexaenoic acid, with Aβ, and their effect on Aβ aggregation has not been explored in detail. Therefore, we have investigated the effect of docosahexaenoic acid, arachidonic acid and the saturated fatty acid arachidic acid on monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography. The polyunsaturated fatty acids docosahexaenoic acid and arachidonic acid at micellar concentrations stabilized soluble Aβ42 wild‐type protofibrils, thereby hindering their conversion to insoluble fibrils. As a consequence, docosahexaenoic acid sustained amyloid‐β‐induced toxicity in PC12 cells over time, whereas Aβ without docosahexaenoic acid stabilization resulted in reduced toxicity, as Aβ formed fibrils. Arachidic acid had no effect on Aβ aggregation, and neither of the fatty acids had any protofibril‐stabilizing effect on Aβ42 harboring the Arctic mutation (AβE22G). Consequently, AβArctic‐induced toxicity could not be sustained using docosahexaenoic acid. These results provide new insights into the toxicity of different Aβ aggregates and how endogenous lipids can affect Aβ aggregation.

Attenuated amyloid-β aggregation and neurotoxicity owing to methionine oxidation

Aggregation of the amyloid-&bgr; (A&bgr;) peptide into amyloid plaques is a characteristic feature of Alzheimers disease neuropathogenesis. We and others have previously demonstrated delayed A&bgr; aggregation as a consequence of oxidizing a single methionine residue at position 35 (Met-35). Here, we examined the consequences of Met-35 oxidation on the extremely aggregation-prone peptides A&bgr;1-42 and A&bgr;1-40Arctic with respect to protofibril and oligomer formation as well as neurotoxicity. Size exclusion chromatography and mass spectrometry demonstrated that monomer/dimers prevailed over larger oligomers after oxidizing Met-35, and consequently protofibril formation and aggregation of both A&bgr;1-42 and A&bgr;1-40Arctic were delayed. The oxidized peptides completely lacked neurotoxic effects in cortical neuronal cultures under these conditions, in contrast to the neurotoxic properties of the unoxidized peptides. We conclude that oxidation of Met-35 significantly attenuates aggregation of A&bgr;1-42 and A&bgr;1-40Arctic, and thereby reduces neurotoxicity.

O4-06-01

ronal development. Methods: We used the powerful method of in utero electroporation of DNA constructs into the developing rodent cortex to either knock down or over-express APP, its proteolytic derivatives, and certain binding partners. In our studies, DNA constructs are injected into one of the lateral ventricles of the embryonic rodent brain, and electroporated into a subset of cells in the cortical ventricular zone. DNA constructs encoding GFP are co-electroporated to follow transfected cells. Postsurgery, embryos develop normally in utero. We examine the effects of APP knock down at 3612and 30-days post electroporation. Results: In utero electroporation of APP shRNA constructs led to the generation of mosaic regions of developing cortex in which APP expressing and nonexpressing cells neighbor one another. Qualitative and quantitative analyses revealed that neuronal precursors in the cortex require APP to migrate correctly into the cortical plate. Cells that received GFP and an inactive APP shRNA migrated properly through the intermediate zone and into the cortical plate, whereas the majority of cells that received active APP shRNA were retarded in the intermediate zone, where they prematurely differentiated into neurons. The minority of cells expressing active shRNA that migrated into the cortical plate did not migrate to the correct layer and displayed abnormal neurite outgrowth. Co-electroporation of holoAPP cDNA substantially rescued the shRNA-mediated migration defect. Various proteolytic derivatives of APP as well as APLP-1 and -2 are being tested for their rescue abilities, to address the mechanism by which APP performs this function. We conclude that acute knock-down of APP in utero reveals a required function for the precursor in cortical cell migration during development.