The β- and γ-secretase-driven cleavage of the amyloid precursor protein (APP) gives rise to the amyloid β peptide, which is believed to be the main driver of neurodegeneration in Alzheimer’s disease (AD). As it is prominently detectable in extracellular plaques in post-mortem AD brain samples, research in recent decades focused on the pathological role of extracellular amyloid β aggregation, widely neglecting the potential meaning of very early generation of amyloid β inside the cell. In the last few years, the importance of intracellular amyloid β (iAβ) as a strong player in neurodegeneration has been indicated by a rising number of studies. In this review, iAβ is highlighted as a crucial APP cleavage fragment, able to manipulate intracellular pathways and foster neurodegeneration. We demonstrate its relevance as a pathological marker and shed light on initial studies aiming to modulate iAβ through pharmacological treatment, which has been shown to have beneficial effects on cognitive properties in animal models. Finally, we display the relevance of viral infections on iAβ generation and point out future directions urgently needed to manifest the potential relevance of iAβ in Alzheimer’s disease.
CRISPR/Cas9 gene editing is a revolutionary method used to study gene function by transcript silencing, knock-out, or activation. The knock-in of DNA fragments to endogenous genes of interest is another promising approach to study molecular pathways but is technically challenging. Many approaches have been suggested, but the proof of correct integration has often been relied on less convenient validation experiments. Within this work, we investigated homology-directed repair (HDR), non-homologous end joining (NHEJ), and PCRextension (PCRext) based approaches as three different methods to knock-in large DNA fragments (>1000 bp), and compared feasibility, cost effectiveness, and reliability. As a knock-in fragment, we used a fluorescent reporter sequence in order to directly assess successful integration by microscopy, subsequently proven by sequencing. For NHEJ and PCRext, we demonstrate that it is insufficient to rely on the fluorescent reporter due to false positive results. Both NHEJ and PCRext failed to reliably knock-in large DNA sequences, they were accompanied by massive generation of InDels driving the methodology cost-intensive and non-reliable. In contrast, combination of CRISPR/Cas9 and HDR revealed correct integration, proven by correct fluorescence of the subcellular localization and sequencing, and thus, corresponds to the method of choice for large fragment integration. Next to HEK293T, we demonstrate successful HDR based knock-in in human induced pluripotent stem cells (hiPSCs). Subsequent differentiation of gene-edited hiPSCs into cerebral organoids showed relevance of the approach to study subcellular protein localization and abundance in 3D tissue.
The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates consisting of its adapter protein FE65, the histone acetyltransferase TIP60 and the tumour suppressor proteins p53 and PML. APP C-terminal (APP-CT50) complexes co-localize and co-precipitate with p53 and PML. The PML nuclear body generation is induced and fusion occurs over time depending on APP signalling and STED imaging revealed active gene expression within the complex. We further show that the nuclear aggregates of APP-CT50 fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimer’s disease brains reveal a highly significant reduction of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT50 signalling to the nucleus takes place in the aged human brain and is involved in the pathophysiology of AD.
Cerebral organoids are a promising model to study human brain function and disease, though the high inter-organoid variability of the mini-brains is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different hiPSC lines, which enables the identification of cells from different origin within a single organoid. The method combines a gene editing workflow and subsequent organoid differentiation and offers a unique tool to study gene function in a complex human 3D tissue-like model. Using a CRISPR/Cas9 gene editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs and subsequently used as a marker to identify each cell line. Mixtures of differently edited cells were seeded to induce embryoid body formation and cerebral organoid differentiation. As a consequence, the development of the 3D tissue was detectable by live confocal fluorescence microscopy and immunofluorescence staining in fixed samples. Analysis of mixed organoids allowed the identification and examination of specifically labeled cells in the organoid that belong to each of the two hiPSC donor lines. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, and thus the mixed organoids overcome the inter-organoid inhomogeneity limitations. The approach aims to pave the way for the reliable analysis of human genetic disorders by the use of organoids and to fundamentally understand the molecular mechanisms underlying pathological conditions.
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