Lars Tjernberg Group
Gamma-secretase and the Amyloid beta-peptide
The polymerization of the amyloid β (Aβ)-peptide into neurotoxic oligomers is a pathogenic event in Alzheimer's disease (AD). This 40-42 residue peptide is released from its precursor, APP, by the action of a multi-transmembrane protease called γ-secretase. Thus, to inhibit Aβ-production by γ-secretase inhibitors, or to stop the formation of neurotoxic Aβ oligomers by oligomerization inhibitors, are potential treatment strategies. Since γ-secretase has a multitude of substrates, one of the most important being Notch, it will be necessary to specifically inhibit APP-processing. Our main aims are to find γ-secretase associated proteins (GSAPs) that affect APP, but not Notch, processing and to find efficient inhibitors of Aβ-oligomerization.
We have developed a highly efficient and selective method for affinity purification of GSAPs in membranes prepared from brain. The purified proteins were identified in a sensitive and unbiased manner by liquid chromatography- tandem mass spectrometry (1). The effect of the GSAPs on Aβ-production was tested by siRNA mediated knockdown of their expression in cell systems. Interestingly, around 50% the GSAPs were found to have a significant effect on Aβ-production. The GSAPs that reduced Aβ-production after siRNA treatment were further studied with respect to their effect on Notch-processing, and several of them showed only a limited effect on Notch. Hence, we have identified around ten novel GSAPs that selectively affect APP-processing, and could be potential drug targets for treatment of AD. At present, we are evaluating the GSAPs with respect to their subcellular co-localization with γ-secretase, their interaction with γ-secretase, and their potential involvement in AD pathology. Based on these studies, we will select a few of them for further studies aimed at investigating how they could be used as drug targets.
In order to find Aβ-oligomerization inhibitors, we have developed a high throughput screen (HTS). From a large compound library, around one thousand potential oligomerization inhibitors were identified by using this HTS. To evaluate these compounds we are now, in collaboration with the Royal Institute of Technology, developing a sensitive on-line, in solution method that gives information on the early stages of the Aβ-oligomerization process.
Previously, we used laser capture microscopy for purifying amyloid plaque cores and showed that they contained only Aβ. Now, we have studied which C-terminal variants of Aβ that are deposited in the AD brain, and found that a longer variant, Aβ43, is frequently deposited (2). This variant is more hydrophobic and potentially more toxic than Aβ42, and could thus be of importance for AD. We are now developing methods for rapid quantification of soluble Aβ43 in brain and cerebrospinal fluid. We have also investigated the intracellular levels of Aβ in pyramidal neurons as well as Purkinje cells, and shown that the levels of the toxic Aβ42 are elevated in pyramidal cells in AD. Thus, the Aβ42 levels are selectively elevated in cells that are at risk in AD (3). Presently, we use laser capture microdissection combined with mass spectrometry to identify proteins that are differently expressed in pyramidal neurons from AD compared to controls.
|Susanne Frykman||Researcher||Read more about Susanne's research|
|Sophia Schedin Weiss||Researcher||Read more about Sophia's research|