Characterization of the Alzheimer's disease-associated clac protein

Abstract

Alzheimer's disease (AD) is the most prevalent form of dementia in elderly people and is neuropathologically characterized by the presence of extracellular senile plaques (SPs) in the brain. SPs are mainly composed of the amyloid beta-peptide (Abeta), proteolytically derived from the amyloid beta-precursor protein by sequential cleavages performed by beta- and gammasecretase, to generate Abeta species of 40 and 42 amino acids, respectively. The mechanism underlying the formation of SPs is largely unknown but it is possible that the less dense Abeta deposits, referred to as diffuse plaques, represent precursors to the SPs that occurs in the brain at a later stage of the disease. In addition to Abeta, a number of other non-Abeta components have been found to co-deposit with SPs. Many studies performed in vitro and in vivo on these so-called plaque-associated proteins suggest that they are able to modulate Abeta aggregation, either by facilitating or preventing Abeta fibril growth. An increased plaque load in the brain may also be caused by Abeta-binding components that increase the persistence of plaque by affecting the clearance. Thus, further characterization of these proteins and their impact on Abeta aggregation is imperative for understanding the events underlying formation of amyloid deposits in vivo.

The work presented in this thesis has focused on one of these plaque-associated proteins, the collagenous Alzheimer amyloid plaque component (CLAC) and its interaction with Abeta. CLAC is generated through furin cleavage of the precursor protein CLAC-P/collagen XXV, a novel transmembrane collagen. In paper I we found that CLAC was the antigen causing the AMY immunoreactivity, which is co-localized with SPs in AD brain. A system for the expression and purification of recombinant CLAC in a mammalian cell line was devised in paper II, which enabled us to study the in vitro properties of CLAC. We showed that CLAC displays features characteristic of a collagen protein, e.g. it forms a partly protease-resistant triple-helical structure, exhibits an intermediate affinity for heparin and is glycosylated. In order to study the molecular interactions between CLAC and Abeta in vitro, we developed a solid-phase binding immunoassay. CLAC was found to bind aggregated Abeta in a concentration-dependent manner and binding was impaired by increasing salt concentrations, suggesting a partly ionic dependent CLAC/Abeta interaction. The LIKRRLIK sequence within non-collagenous domain 2 of CLAC was identified as an Abeta-binding motif. Deletion or substitution of this motif generated CLAC variants with diminished Abeta-binding affinity. In paper III, sequences in Abeta involved in CLAC-binding were examined. The central region of Abeta was shown to be necessary and sufficient for binding to CLAC and the binding correlated better with the aggregation state of the peptide, than with the primary sequence. This suggests that CLAC has low sequence specificity and is in agreement with observations presented in paper II, which show that CLAC is able to bind other fibrils formed from non-amyloid-beta component and amylin.

Understanding the role of CLAC/Abeta interaction in AD is essential for future research aiming at interacting with the CLAC/Abeta binding. In paper III the effect of CLAC on Abeta fibril elongation was examined using surface plasmon resonance spectroscopy. Treatment of Abeta fibrils with CLAC reduced the rate of Abeta fibril elongation by 20 to 30%. The effect of CLAC on Abeta fibrils was further studied in paper IV using assays based on turbidity, Thioflavin T binding, sedimentation analysis and electron microscopy. Incubation of preformed fibrils with CLAC led to the formation of larger aggregates compared to non CLAC-treated samples. Finally, we investigated whether the CLAC-induced fibrils could be of some biological relevance by a protease resistance assay. Abeta fibrils incubated with CLAC were more resistant to proteolytic degradation and it is possible that CLAC may act in a similar way in vivo. We suggest that CLAC effects AD pathogenesis by binding and assembling Abeta aggregates and thereby altering the turnover and persistence of SPs in AD brain.