Amyloid Precursor Protein dependent generation of nuclear complexes in gene edited iPSC derived neurons

• Applicant: Privatdozent Dr. Thorsten Müller
• Subject Area: Experimental Models for the Understanding of Nervous System Diseases ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 270451600

The amyloid precursor protein (APP) is a central player in the pathophysiology of Alzheimer’s disease. The intracellular domain of APP is involved in a signal transduction pathway with yet less known function. As a consequence of the APP cleavage or phosphorylation changes, the adapter protein FE65, which is initially tethered to the cell membrane via its interaction to the intracellular domain of APP, is capable to translocate to the nucleus and to set up a multimeric complex consisting of additional proteins, which have function in DNA repair or replication mechanisms. The complex is highly dynamic including the initial generation of small aggregates, their fusion to larger structures of up to 2µm in size with the final consequence of cell death. Within this project we intend to study the APP signal transduction pathway by using recent developments in stem cell biology, gene editing, and confocal imaging techniques. We aim to unravel the specific factors causing translocation of FE65 to the nucleus. We will assess the function of the complex in neurons and their meaning for the Alzheimer pathophysiology. In more detail, the project is dedicated to establish a human inducible pluripotent stem cell line, in which FE65 is fused to a fluorescent protein, enabling the analysis of its subcellular localization by microscopic techniques. Studies will be performed in neurons differentiated from the edited stem cell line. Additionally, the FE65 gene editing workflow will be applied to already existing mutant APP stem cell lines to study the potential impact of familiar APP mutations on FE65 translocation in a non-overexpressing neuronal model corresponding to the best “low artificial” cell system available to date. Composition of FE65 related nuclear complexes will be encouraged including a set of new candidate proteins identified by our lab in preliminary work to better understand the functional impact of the nuclear aggregates. Relevance of the complexes for the nuclear integrity will be studied in neuronal cells differentiated from iPS cells edited for FE65 as well as for Lamin B, which will be tagged by another fluorescent protein. Modifications of the nuclear envelope, summarized under the term laminopathy, are currently discussed to be an early modification within the pathophysiology of Alzheimer’s disease. As first results of our group pointed to nuclear envelope invaginations in FE65 dependent complex over-expressing cells, the aimed studies within the new proposal have the potential to identify an APP/FE65 dependent signal transduction pathway with central relevance for Alzheimer’s disease and might unravel the mechanism behind the observed laminopathy phenotype and the function of the nuclear aggregates. Our intended experiments are finally completed by immune-histological studies of complex relevant proteins in the human hippocampus of patients and controls.

• DFG Programme: Research Grants