Inhibition of β-amyloid production by APP ligands

Projektdetails:

Projektbeschreibung

The overall aim of the project is to understand how the interaction between divalent metal-ions and the amyloid precursor protein (APP), other members of the APP family and Aß is involved in the genesis of Alzheimer's disease (AD). This will provide vital fundamental information and allow rational therapeutic approaches to be devised. We discovered that APP binds zinc(Il) and copper(Il) at two distinct sites. In the APP gene family, both, the binding site for zinc(Il) and copper(Il) are conserved in APLP2, whereas in APLP1 only the zinc(Il) site is present. Whereas Zn(II) is assumed to play a purely structural role, our studies on copper binding revealed that APP has an in vilro function in electron transfer to Cu(II) leading to oxidative modification of APP. Recently, we found copper to impair the levels of Aß in transfected copper-resistanct ells. Additionally, we have shown that APP expressionc an mediate Cu-toxicity in primary neurons.

The effects of these interactions are likely to be important for the role of APP and Aß in Alzheimer's disease. Thus, our current understanding is that copper and/or zinc binding is central to the normal cellular function of APP and is also important in pathogenic mechanisms in Alzheimer's disease. We therefore want to identify agonists of APP ligand binding sites (e.g. of the copper-binding site) that are able to inhibit amyloidogenic proteolytic processing of APP. Lead compounds will be discovered followed by a further screen for small APP ligands. These are internalized as APP complexes and thus can cross the cell membrane. Their properties of anti-amyloid drugs will be investigated in a cell culture system.

Specific Aim 1) To understand the basic mechanism underlying the interaction between divalent metal-ions and APP, other members of the APP family and how Aß production and release is influenced.

Specific Aim 2) To identify if the copper/zinc binding sites of APP can represent potential novel drug target sites and agonists of these ligand binding sites of APP might be beneficial for the treatment of Alzheimer's disease.

We propose a research program combining biochemical, immunohistochemical and cellular approaches to start a riskbenefit analysis of divalent metal binding agonists of APP in vitro.

Abschlussbericht

Alzheimer's disease is the most common form of dementia affecting elderly people. In all forms of the disease accumulating evidence supports the hypothesis that cerebral amyloid Aß peptide deposition is the essential pathogenic event. The major component of amyloid plaque proteins, Aß, is derived from the transmembrane amyloid precursor protein (APP). This peptide is generated by two as yet unidentified protease activities (termed ß- and y-secretases).

Alternatively, APP can be cleaved by a third activity (α-secretase) within the Aß domain and thus precludes the generation of Aß. 

Interestingly, mutations in three different genes (APP, Presenilin 1 and Presenilin 2) were shown to cause genetically inherited forms of the disease, all influencing APP processing to the generation of Aß. It has been commonly accepted that an inhibition of amyloidogenic cleavage of APP would directly lead to a reduced risk for amyloid plaque formation and thus for developing the disease. For instance, small ligand agonists, e.g. agonist for the copper-binding site of APR could easily enter the cell by APP internalization as APP-complexes and inhibit APP transport to the cellular compartment where amyloidogenic cleavage of APP takes place. A resulting reduced amyloid Aß production is expected to slow down tlre progression of the disease and directly targets the pathological hallmark of Alzheimer's disease, the amyloid plaque formation.

We have successfully started a novel approach to directly influence the pathological processing of APP in a relevant pharmacological manner by mimicking APP ligand functions that directly attack amyloid Aß generation. The chosen target site is in the N-terminal domain of APP that contains two metal binding motifs. Both zinc and copper are bound with a 1:1 stoichiometry at the two independent sites. The copper binding site is similar to the type 2 copper binding motif found in other copper binding proteins. Our published data suggest that APP is involved in the homeostatic control of copper in the brain and in the liver in vivo. On the cellular level, APP possesses homeostatic mechanisms to assist in the maintenance of the correct concentrations of copper. Copper is a trace element essential for normal cell homeostasis and the intracellular trafficking of this metal to copper-dependent proteins is fundamental to normal cellular metabolism. The major physiological role of copper is to serve as a cofactor to a number of key metabolic enzymes.

Based on our published data, our novel findings show that, in addition to its general role as a cofactor, copper can regulate the amyloidogenic pathway of APP by inducing the nonamyloidogenic processing of APP through specific binding to the copper binding motif of APP. Analysis of copper-induced conformational changes in the amino-terminal domain indicates that it represents a most accessible site and both secondary and tertiary structure changes take place upon copper binding. These copper-induced conformational changes could play an important role in the normal function of APP in vivo. We have expressed and characterized this APP domain with the copper binding site and analyzed functional and conformational consequences of copper binding for several APP family members. In addition to providing important insights on copper binding, these results confirmed our assumption that the copper binding site of APP can represent a potential drug target site.

Thus, our proof-of-principle study already reveals that the copper binding site can be used to identify compounds as leads for use as a secretase inhibitor alternative in a therapeutic approach. Since redox reactivity of copper leads to risks of damage to cell and tissues and to minimize negative effects, we will proceed with a search for copper agonists.