Identifizierung und Charakterisierung eines spezifischen Liganden für das Alzheimer Amyloid$\beta$-Peptid (A$\beta$)

Abstract

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder. Extracellular amyloid plaques in the brain are one of the main histopathological hallmarks of this disease. The amyloid-cascade-hypothesis" assigns the amyloid-$\beta$-peptide (A$\beta$) a central role in the pathogenesis of AD. lt is assumed that progressive accumulation of A$\beta$ initiates a complex multicellular cascade that includes neuritic dystrophy, microgliosis, astrocytosis, neuronal dysfunction and loss of synapsis. The consequences ofthis multicellular cascade are impaired amnestic and cognitive functions of patients suffered by AD. Based an this hypothesis, diagnostic and therapeutic strategies targeting A$\beta$ and A$\beta$ fibrils are being pursued. In the present dissertation peptides consisting of D-amino acids (D-peptides) should be identified that bind to A$\beta$ with high specifity. D-peptides are thought to be protease-resistant and less immunogenic than the respective L-enantimer (Schumacher et al., 1996). A screening of a randomized 12mer peptide library presented an bacteriophage M13 for binding to A$\beta_{l-42}$ consisting of D-amino acids as target was performed. After four rounds of selection and amplification different peptides were selected. The amino acid sequence of Q S H Y R H I S P A Q V (all amino acids in one-letter-code) was obtained in 20 of 39 selected peptides. The mirror image of this dominating peptide (D-peptide) binds to synthetic A$\beta_{1-42}$ consisting of L-amino acids as shown by fluorescence spectroscopy titration experiments. An estimation of the dissociation equilibrium constant (KD) with non-linear regression produced a value in the micromolar range. Binding of D-Pepl to natural A$\beta$ was verified through in-vitro-targeting of A$\beta$ precipitations in tissue sections derived from patients that suffered from AD. Amyloid precipitations in tissue sections of patients that suffered from other amyloidosis disorders were not marked by D-Pepl. In cytotoxicity tests, it has been demonstrated that the D-peptide has a positive effect an the cytotoxicity of A$\beta$. The influence depends an the concentration of the D-peptide and the fibril formation state of A$\beta$. The results of the cytotoxicity assays could not be explained based an the nucleations-elongations-modell by Lansbury et al. (consisting of two steps: building of a aggregation nucleus and elongation of fibrils) (Jarrett et al., 1993 ; Jarrett and Lansbury, 1993; Harper and Lansbury, 1997). An interpretation of all results of the cytotoxicity tests becomes possible by consideration of an extended model by Harper et al. which adjuLansbury, 1993; Harper and Lansbury, 1997). An interpretation of all results of the cytotoxicity tests becomes possible by consideration of an extended model by Harper et al.. which adjusts protofilaments (Harper et al., 1999). Thereby an influence of D-Pepl to the aggregation of ALansbury, 1993; Harper and Lansbury, 1997). An interpretation of all results of the cytotoxicity tests becomes possible by consideration of an extended model by Harper et al.. which adjusts protofilaments (Harper et al., 1999). Thereby an influence of D-Pepl to the aggregation of $\beta$ as well as different mechanisms for toxicity of protofilaments and fibrils was assumed