Soluble intermediates of the amyloid-β (Aβ) aggregation process are suggested to play a central role in the pathogenesis of Alzheimer’s disease (AD) by causing synaptic dysfunction and neuronal loss. In this thesis, soluble Aβ aggregates have been studied with a particular focus on the Aβ protofibril, which has served as the antigen for developing conformation dependent monoclonal antibodies. Antibodies generated from mice immunized with Aβ protofibrils were characterized regarding Aβ binding properties and the amino acid sequences of their antigen binding sites. A conformation dependent IgG antibody, mAb158, was further characterized and found to bind to Aβ protofibrils with a 200-fold higher affinity than to monomeric Aβ without affinity for soluble amyloid-β precursor protein (AβPP) or other amyloidogenic proteins. A sandwich enzyme-linked immunosorbent assay (ELISA) based on mAb158 was used to measure soluble Aβ protofibrils in brain extracts from AβPP-transgenic mice. Low levels of protofibrils could also be detected in human AD brain. However, positive signals generated from measurements in AD and control CSF samples were attributed to interference from heterophilic antibodies (HA), generating false positive signals by cross-binding the assay antibodies; consequently, a study on HA interference in Aβ oligomer ELISAs was initiated. A large set of plasma and CSF samples from AD and non-AD subjects were analyzed with and without measures taken to block HA interference, revealing that virtually all signals above the assay limit of detection were false and generated by HA interference. Many types of soluble Aβ aggregates have been described and suggested to impair neuron and synapse function. To investigate the soluble Aβ pool, synthetic Aβ and brain extracts from AβPP-transgenic mice and AD patients were ultracentrifuged on a density gradient to separate Aβ by size under native conditions. Four distinct gradient fractions were defined based on the appearance of synthetic Aβ in atomic force microscopy (AFM) and immunoreactivity in our protofibril specific sandwich ELISA. Interestingly, most Aβ from AD patients and AβPP-transgenic mice separated in the same fraction as toxic synthetic protofibrils.

The present study assessed olfactory and cognitive abilities in two strains of Alzheimer’s disease (AD) model mice and in healthy control mice over a four month time period. To this end an operant conditioning paradigm using an automated olfactometer and a spatial learning test with non-olfactory cues were employed and data on olfactory learning and memory, discrimination, and sensitivity as well as spatial learning and memory were collected. The mice were between 6 to 7 month old at the beginning of the study and 9 to 10 months old at the end of the data collection, that is, in the age range when the animals are supposed to display marked neuroanatomical changes typical of AD. The results demonstrate that there were no systematic differences in olfactory performance and spatial learning and memory abilities of AD model mice and the control mice up to the age they were tested. Further, there was no indication of an age-related decline in performance in any of the mouse strains across the testing period. Several reasons might account for the observed lack of difference in olfactory and cognitive performance between the mouse strains tested here: the AD model mice might not develop amyloid plaques and neurofibrillary tangles at all or they might develop them later than stated by the supplier. Alternatively, the AD model mice may have developed AD-typical neuroanatomical changes but these do not, or not yet, affect their olfactory performance and/or spatial learning and memory capabilities. Ongoing data collection will help to evaluate which of these explanations holds true.

Prion is an infectious isoform of a normal cellular protein which is capable of converting the non-prion form of the same protein into the alternative prion form. Mammalian prion protein PrP is responsible for prion formation in mammals, causing a series of fatal and incurable prion diseases. (1) We constructed, for the first time, a two-component system to phenotypically monitor the conformational status of PrP in the yeast cells. In this system, the prion domain of Sup35 (Sup35N) was fused to PrP90-230, and the initial formation of the PrPSc-like conformation stimulated prion formation of Sup35N, which in turn converted soluble Sup35 into the prion isoform, leading to a detectable phenotype. Prion-like properties of PrP were studied in this novel yeast model system. Additionally, we employed this system to study amyloidogenic protein Aβ42 aggregation in the yeast model. It has been suggested that the ability to form transmissible amyloids (prions) is widespread among yeast proteins and is likely intrinsic to proteins from other organisms. However, the distribution of yeast prions in natural conditions is not yet clear, which prevents us from understanding the relationship between prions and their adaptive roles in various environmental conditions. (2) We modified and developed sequence and phenotype-independent approaches for prion detection and monitoring. We employed these approaches for prion-profiling among yeast strains of various origins. (3) Lastly, we found a prion-like state [MCS+] causing nonsense suppression in the absence of the Sup35 prion domain. Our results suggested that [MCS+] is determined by both a prion factor and a nuclear factor. The prion-related properties of [MCS+] were studied by genetic and biochemical approaches.

We have recently established that aberrant alkylacylglycerophosphocholine metabolism results in the increased tissue concentration of platelet activating factors (PAFs) in the temporal cortex of Alzheimer Disease (AD) patients and in TgCRND8 mice over-expressing mutant human amyloid precursor protein. PAF lipids activate a G-protein coupled receptor (PAFR) reported to be expressed by microglia and subsets of neurons in rat. It is not known whether this same expression pattern is recapitulated in mice however, as the expression has only been inferred by use of pharmacological PAFR antagonists, many of which impact on both PAFR-dependent and PAFR-independent signalling pathways. PAFR plays a role in long term potentiation (LTP) induction in rats. PAFR has also been implicated in behavioural indices of spatial learning and memory in rats. Contradictory reports using mice provide ambiguity regarding the role of PAFR in LTP induction in mice. To assess whether PAFR is expressed in murine neurons, I localized PAFR mRNA in wild-type C57BL/6 mice using PAFR KO mice as a negative control. I further showed that the loss of PAFR did not impair learning and memory although this assessment must be considered preliminary as the behavioural test employed was not optimized to detect changes in learning and memory of C57BL/6 mice over time adequately.Finally, I showed that the loss of PAFR in TgCRND8 mouse model of AD had no impact upon Aβ plaque number. My observations suggest that PAFR is restricted to microglial-like cells in mouse hippocampus and as such, it may not play a role in learning and memory.

We have recently established that aberrant alkylacylglycerophosphocholine metabolism results in the increased tissue concentration of platelet activating factors (PAFs) in the temporal cortex of Alzheimer Disease (AD) patients and in TgCRND8 mice over-expressing mutant human amyloid precursor protein. PAF lipids activate a G-protein coupled receptor (PAFR) reported to be expressed by microglia and subsets of neurons in rat. It is not known whether this same expression pattern is recapitulated in mice however, as the expression has only been inferred by use of pharmacological PAFR antagonists, many of which impact on both PAFR-dependent and PAFR-independent signalling pathways. PAFR plays a role in long term potentiation (LTP) induction in rats. PAFR has also been implicated in behavioural indices of spatial learning and memory in rats. Contradictory reports using mice provide ambiguity regarding the role of PAFR in LTP induction in mice. To assess whether PAFR is expressed in murine neurons, I localized PAFR mRNA in wild-type C57BL/6 mice using PAFR KO mice as a negative control. I further showed that the loss of PAFR did not impair learning and memory although this assessment must be considered preliminary as the behavioural test employed was not optimized to detect changes in learning and memory of C57BL/6 mice over time adequately.Finally, I showed that the loss of PAFR in TgCRND8 mouse model of AD had no impact upon Aβ plaque number. My observations suggest that PAFR is restricted to microglial-like cells in mouse hippocampus and as such, it may not play a role in learning and memory.

A 20 weeks project at Karolinska Institutet (KI), Huddinge, Sweden is in this master thesis summarized. Alzheimer’s disease is the most common form of dementia in the world. One of the pathological hallmarks seen in AD patients consists of amyloid plaques assembled of beta amyloid (Aβ) peptide aggregates. A lot of research has been done on Aβ40 and Aβ42 but not on the longer variant with 43 residues. An earlier study by Welander et al, quantified the Aβ43 peptide from amyloid plaque cores with high-performance liquid chromatography coupled to mass-spectrometry (HPLC-MS/MS)1. Here, I present the initial development of an enzyme-linked immunosorbent assay (ELISA) with the goal to quantify Aβ43 peptides in soluble fractions of human brain tissue. An ELISA method with the possibility to quantify Aβ43 peptides from cerebral spinal fluid might have the prospect to serve as a diagnostic tool for AD in the future. Commercial ELISA kits coated with antibodies against all Aβ species was not suitable for detecting Aβ43 in soluble brain tissue from human AD patients. This is due to the high amount of Aβ40 (and in some extent Aβ42) in the samples, which will bind to the same epitope as Aβ43 on the capturing antibody. These shorter Aβ species will be in excess and bind to the capturing antibody thereby ousting Aβ43 from binding in. A better way for quantifying Aβ43 with ELISA might instead be to coat a polystyrene plate with α-Aβ43 antibodies, which are c-terminal specific to Aβ43. This will abolish the competition between the different Aβ species and function as an immunoprecipitation of unwanted species. This yielded adequate quantification of Aβ43 (2.64 pM) from tris-buffer saline (TBS) fractions from a human brain sample from AD.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 150-191).

Thesis (Ph.D.)--University of Central Florida, 2008. / Adviser: Sic L. Chan. Includes bibliographical references (p. 74-84).

Thesis (Ph.D.)--University of Central Florida, 2009. / Adviser: Kiminobu Sugaya. Includes bibliographical references (p. 45-50).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 114-145).