Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells

Abstract

Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase processing in cells is not fully understood. Structural studies of PS/γ-secretase enfolding a fragment of APP or Notch1 showed that the two substrates engage the protease in broadly similar ways, suggesting the limited substrate specificity of PS/γ-secretase. In the present study, we developed a new multiplexed imaging platform that, for the first time, allowed us to quantitatively monitor how PS/γ-secretase processes two different substrates (e.g., APP vs. Notch1) in the same cell. In this assay, we utilized the recently reported, spectrally compatible visible and near-infrared (NIR)-range Förster resonance energy transfer (FRET) biosensors that permit quantitative recording of PS/γ-secretase activity in live cells. Here, we show that, overall, PS/γ-secretase similarly cleaves Notch1 N100, wild-type APP C99, and familial Alzheimer’s disease (FAD)-linked APP C99 mutants in Chinese hamster ovary (CHO) cells, which further supports the limited PS/γ-secretase substrate specificity. On the other hand, a cell-by-cell basis analysis demonstrates a certain degree of variability in substrate recognition and processing by PS/γ-secretase among different cells. Our new multiplexed FRET assay could be a useful tool to better understand how PS/γ-secretase processes its multiple substrates in normal and disease conditions in live, intact cells.

In-Depth Characterization of Endo-Lysosomal Aβ in Intact Neurons

“…The selective detection of intracellular Aβ is challenging because antibodies against Aβ cross-react with the Aβ precursor fragments. Our recently developed genetically encoded Förster resonance energy transfer (FRET)-based C99 720-670 biosensor [ 23 , 33 , 34 ] has enabled us to uncover that C99 is predominantly processed by endogenous γ-secretase in late endosomes and lysosomes in intact/live neurons [ 23 ]. To develop the C99 720-670 biosensor, we first tagged the human C99 (APP-CTFβ) containing the APP signal peptide sequence with miRFP720 [ 35 ].…”

“…However, the detailed properties of intracellular Aβ are not fully established. We have recently developed APP C99-based FRET biosensors that enable the quantitative recording of C99 processing by γ-secretase in live neurons [ 23 , 31 , 33 , 34 ]. Combining these novel reporter probes with high-resolution confocal microscopy, we successfully detected predominant γ-secretase activity in late endosomes and lysosomes in intact neurons [ 23 ].…”

Recording γ-secretase activity in living mouse brains

“…In the same line, our FRET-based biosensors have allowed “visualizing” that γ-secretase activity is heterogeneously regulated on a cell-by-cell basis in primary neurons (Maesako et al, 2020). Furthermore, the cell-by-cell heterogeneity in γ-secretase activity was further verified using 1) unique multiplexing FRET analysis in which the processing of two different substrates (e.g., APP vs. Notch1) by γ-secretase can be simultaneously measured in the same cell (Houser et al, 2021), and 2) multiplexed immunocytochemistry in which intracellular Aβ is detected on a cell-by-cell basis (McKendell et al, 2022). Our new study further adds that each neuron exhibits a distinct level of γ-secretase activity in intact mouse brains.…”

“…P < 0.05 was considered statistically significant. The sample size was calculated based on previous correlation analysis in our in vitro studies (Houser et al, 2021; Maesako et al, 2022) and was estimated to include approximately n = 200 - 250 neurons/region-of-interests (ROIs) per animal. Pseudo-color images corresponding to the 720/670 ratios were generated in MATLAB (MathWorks, Natick, MA).…”